WO2024134502A1 - Engineered double-strand rna ligases and uses thereof - Google Patents

Engineered double-strand rna ligases and uses thereof Download PDF

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WO2024134502A1
WO2024134502A1 PCT/IB2023/062949 IB2023062949W WO2024134502A1 WO 2024134502 A1 WO2024134502 A1 WO 2024134502A1 IB 2023062949 W IB2023062949 W IB 2023062949W WO 2024134502 A1 WO2024134502 A1 WO 2024134502A1
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amino acid
seq
acid sequence
engineered
oligonucleotide
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PCT/IB2023/062949
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French (fr)
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Gregory Mann
Frederic Valentin STANGER
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Novartis Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes

Definitions

  • the present disclosure relates to the field of biotechnology, in particular to engineered double-stranded RNA (dsRNA) ligases and their application in industrial biocatalysis.
  • the present disclosure also relates to a process of producing an engineered dsRNA ligase, and to a method for producing an oligonucleotide by contacting oligonucleotide fragments with an engineered dsRNA ligase.
  • dsRNA double-stranded RNA
  • Therapeutic oligonucleotides including small interfering RNA (siRNA) and inhibitory antisense oligonucleotides (ASOs) have the potential to treat a diverse range of life-threatening diseases.
  • small interfering RNA siRNA
  • ASOs inhibitory antisense oligonucleotides
  • biocatalysis is being more frequently applied in the manufacture of active pharmaceutical ingredients (APIs) since enzymes are capable of highly selective transformation under mild reaction conditions and in aqueous media (Mann, G. & Stanger, F. V. Chimia (Aarau) 74, 407-417 (2020)).
  • APIs active pharmaceutical ingredients
  • the biocatalysis of short oligonucleotide fragments offers a sustainable and economical alternative to the solid phase chemical synthesis of full- length therapeutic oligonucleotides currently used.
  • Shorter oligonucleotides can be synthesized more easily and with higher purities than longer oligonucleotides, simplifying downstream processing and reducing solvent waste. These short oligonucleotide fragments can then be combined using nucleic acid ligases to produce oligonucleotide products. Nucleic acid ligases have shown remarkable tolerance towards unnatural DNA/RNA containing pharmaceutically relevant chemical modifications (Kestemont, D., Herdewijn, P. & Renders, M. Curr Protoc Chem Biol 11, e62 (2019); Kestemont, D. et al. Chemical Communications 54, 6408-6411 (2016); and Nandakumar, J. & Shuman, S.
  • dsRNA ligase to synthesize an siRNA product, starting from short fragments ( ⁇ 9 nts), containing extensive chemical modification, including 2’-OMe, 2’-F modified nucleotides, phosphorothioate backbone modified nucleotides and a terminal fragment that is functionalized with a bulky N- acetyl galactosamine (GalNAc) moiety has previously been described (Mann, G. et al. Tetrahedron Letters 93, 153696 (2022)).
  • GalNAc bulky N- acetyl galactosamine
  • the sequences of oligonucleotides 2, 3 and 5-12 are provided in Table 1.
  • FIG. 3 Comparative data showing the relative peak area % of siRNA (1) present in the reaction samples comprising different concentrations of wild-type enzyme (SEQ ID NO: 2) and engineered enzymes (SEQ ID NOs: 288 and 632) following pre-incubation of the enzyme at 4 °C or 37 °C for 4 h.
  • Enzyme concentration is provided as g/L of shake-flask powder (SFP) produced by lyophilization of frozen clarified lysate as described in the Examples.
  • SFP shake-flask powder
  • the present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides.
  • dsRNA double-stranded RNA
  • the present disclosure also provides gene sequences of engineered polypeptides, recombinant expression vectors comprising the genes, engineered host strains and efficient methods for the production thereof, as well as reaction processes for the biocatalysis of oligonucleotides using engineered polypeptides.
  • the engineered double -stranded RNA (dsRNA) ligase polypeptides described herein have improved catalytic activity as compared to the wild-type dsRNA ligase from which they are derived.
  • the engineered polypeptides provided herein were derived from a wildtype dsRNA ligase from Bacteriophage RB69.
  • the wild-type dsRNA ligase consists of 332 amino acids and has the amino acid sequence shown in SEQ ID NO: 302 (also accessible under accession number Q7Y4V8 in UniProt).
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
  • dsRNA double-stranded RNA
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • engineered dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
  • dsRNA double-stranded RNA
  • engineered dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • dsRNA double-stranded RNA
  • ligase polypeptide which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
  • a polypeptide having dsRNA ligase activity which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • dsRNA double-stranded RNA
  • ligase polypeptide which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
  • a polypeptide having dsRNA ligase activity which comprises an amino acid sequence having
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
  • dsRNA double-stranded RNA
  • the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E ; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q,
  • the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 666. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666.
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • dsRNA double-stranded RNA
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 666; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 370; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.
  • dsRNA double-stranded RNA
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 488; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, or all 3) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.
  • dsRNA double-stranded RNA
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 526; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.
  • dsRNA double-stranded RNA
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 578; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 588 or 590; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • X15 is D or E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 592; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666 and 668. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 642, 646, 664, and 666.
  • the disclosure also provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the ligation reaction conditions include about 1 pM to about 10 mM oligonucleotide fragment, a source of ATP, about 5 mM to about 100 mM divalent cation, and about 0.5 g/L to about 10 g/L engineered dsRNA ligase polypeptide, pH of about 4.0 to about 8.0, and temperature of about 10 °C to about 50°C.
  • the source of ATP comprises ATP, optionally a stoichiometric excess of ATP.
  • the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237,
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218, X221, X228, X
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X; X
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y,
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, or all 4) amino acid residues selected from: X36, X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, or all 3) amino acid residues selected from: X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, or all 3) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, or all 4) amino acid residues selected from: X39, X218, X221 and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • X15 is E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) amino acid residues selected from: X19, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10) amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g.
  • X15 is D; X39 is A; X 53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • the engineered dsRNA ligase polypeptide comprises a purification tag.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
  • 294, 296, 298, 300 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628,
  • the disclosure also provides a polypeptide immobilized on a solid material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide described herein.
  • the disclosure also provides a polynucleotide encoding the engineered dsRNA ligase polypeptide described herein.
  • the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
  • the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667.
  • the polynucleotide comprises a nucleic acid sequence selected from: (a) SEQ ID NOs: 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365,
  • the disclosure also provides an expression vector comprising the polynucleotide described herein.
  • the vector comprises a plasmid, a cosmid, a bacteriophage or a viral vector.
  • the disclosure also provides a host cell comprising the polynucleotide described herein or the expression vector described herein.
  • the host cell is E. coli.
  • the disclosure also provides a method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing the host cell described herein and obtaining an engineered dsRNA ligase polypeptide from the culture.
  • the disclosure also provides an engineered dsRNA ligase catalyst obtainable by culturing the host cells described herein, or according to the method described herein, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cell, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cell, an extract thereof, or isolated product of the extract.
  • the source of ATP comprises ATP.
  • the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
  • the PPK is selected from PPK 12 or ajPAP.
  • the method is performed using a sub-stoichiometric concentration of AMP and/or ATP.
  • the polyphosphate is a polyphosphate salt.
  • the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
  • the divalent cation cofactor is Mg 2+ or Mn 2+ .
  • the method is performed with a divalent cation concentration of 5-100 mM, optionally 30-50 mM.
  • the method further comprises a step of purifying the oligonucleotide.
  • the disclosure also provides use of the engineered dsRNA ligase polypeptide described herein in the production of an oligonucleotide from two or more oligonucleotide fragments.
  • each of the oligonucleotide fragments are 4-16 nucleotides in length, optionally 6-9 nucleotides in length.
  • one or more of the oligonucleotide fragment(s) comprises one or two overhangs.
  • one or more of the oligonucleotide fragments comprises a chemical modification.
  • the chemical modification is selected from: (a) a modified backbone, optionally selected from a phosphorothioate (e.g.
  • a modified nucleotide optionally selected from 2'-O-methyl (2’-OMe), 2'-flouro (2’-F), 2'-deoxy, 2'-deoxy-2’- fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g.
  • LNA locked nucleic acid
  • GAA glycol nucleic acid
  • phosphoramidate e.g.
  • ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives.
  • GalNAc N-Acetylgalactosamine
  • the disclosure also provides a composition
  • a composition comprising: i. the engineered dsRNA ligase polypeptide described herein; ii. a source of ATP; and iii. a divalent cation.
  • the composition further comprises two or more oligonucleotide fragments.
  • the disclosure also provides a kit comprising: i. the engineered dsRNA ligase polypeptide described herein; ii. a source of ATP; iii. a divalent cation; and iv. instructions for use in a method of producing an oligonucleotide from two or more oligonucleotide fragments.
  • the source of ATP comprises ATP.
  • the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
  • the PPK is selected from PPK 12 or ajPAP.
  • the polyphosphate is a polyphosphate salt.
  • the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
  • the divalent cation cofactor is Mg 2+ or Mn 2+ .
  • articles such as “a” and “an” refer to one or more than one (at least one) of the grammatical object of the article.
  • the term “about” typically refers to the value which immediately follows the term ‘about’.
  • “about 15 or more nucleotides” typically refers to 15 or more nucleotides.
  • the term “about” embraces values which are +/- 1, 2 or 3 of the stated value.
  • “about 15 or more nucleotides” may refer to 15+/-3 nucleotides, e.g. 12, 13, 14, 15, 16, 17 or 18 nucleotides.
  • double-stranded RNA ligase” and “dsRNA ligase” are used interchangeably herein to refer to an enzyme having dsRNA ligase activity.
  • a dsRNA ligase polypeptide may also be referred to herein as a “dsRNA ligase catalyst”.
  • a dsRNA ligase of the invention is an ATP-dependent nucleic acid ligase.
  • dsRNA ligase activity typically involves the ATP-dependent formation of a covalent bond between the 3 ’-OH of a ribonucleotide and the 5’-PO4 of a ribonucleotide or deoxyribonucleotide via the following steps: (1) dsRNA ligase reacts with ATP to form a covalent dsRNA ligase-AMP intermediate and release pyrophosphate; (2) AMP is transferred from the dsRNA ligase-AMP intermediate to the 5 ’-phosphate of a 3’ oligonucleotide fragment forming an adenylated oligonucleotide intermediate; and (3) the 3 ’-OH of a 5’ oligonucleotide fragment attacks the 5 ’ phosphate of the adenylated intermediate resulting in the formation of a phosphodiester bond
  • the stoichiometric concentration of cofactor is the theoretical concentration required to achieve complete ligation in a given ligation reaction.
  • the skilled person can readily derive the stoichiometric concentration of ATP required to achieve complete ligation based on the concentration of oligonucleotide fragments and the number of ligation reactions required to produce the oligonucleotide product. For example, a ligation reaction using 1 mM substrate which requires four ligation reactions has a stoichiometric ATP concentration of 4 mM. A stoichiometric excess of ATP can help ensure that complete ligation is achieved.
  • the stoichiometric excess is at least 105% of the theoretical stoichiometric concentration of ATP required to achieve complete ligation, e.g. at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.
  • engineered dsRNA ligase engineered dsRNA ligase polypeptide
  • improved dsRNA ligase polypeptide engineered polypeptide
  • oligonucleotide refers to a nucleic acid, typically comprising up to 100 nucleotides.
  • oligonucleotide product refers to an oligonucleotide formed by the ligation of two or more oligonucleotide fragments by a dsDNA ligase described herein. Oligonucleotide products are also referred to herein simply as oligonucleotides. It will be understood that oligonucleotide products described herein comprise RNA. It will also be understood that oligonucleotide products described herein comprise a double-stranded region.
  • oligonucleotide products described herein comprise RNA and DNA.
  • a portion of the oligonucleotide product may be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.
  • RNAi RNA interference
  • ASO antisense oligonucleotides
  • RNAi is a post-transcriptional, targeted gene-silencing technique that uses RNAi agents to degrade messenger RNA (mRNA) containing the same sequence as the RNAi agent.
  • ASOs are single-stranded nucleic acids that can be used to target mRNA derived from a gene of interest. ASOs can alter gene expression via a number of mechanisms including direct steric blockage of mRNA and ribonuclease H (RNase H) mediated degradation of mRNA.
  • RNAi agents include, as non-limiting examples, siRNAs (small interfering RNAs), dsRNAs (double-stranded RNAs), shRNAs (short hairpin RNAs) and miRNAs (micro RNAs).
  • RNAi agents also include, as additional non-limiting examples, locked nucleic acid (LNA), Morpholino, UNA, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA) and fluoro-arabinonucleic acid (FANA).
  • RNAi agents also include molecules in which one or more strands are a mixture of RNA, DNA, LNA, Morpholino, UNA (unlocked nucleic acid), TNA, GNA, and/or FANA.
  • one or both strands of an RNAi agent could be, for example, RNA, except that one or more RNA nucleotides is replaced by DNA, LNA, Morpholino, UNA, TNA, GNA, and/or FANA, etc.
  • one or both strands of the RNAi agent can be nicked, and both strands can be the same length, or one strand can be shorter than the other.
  • the oligonucleotide of the invention may be any of the RNAi agents described herein.
  • oligonucleotide fragment refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide (or oligonucleotide product). Each oligonucleotide fragment corresponds to a portion of the oligonucleotide product. Oligonucleotide fragments may be referred to herein as “substrates” of the ligation reaction.
  • dsRNA ligase activity involves the ligation of a 5’ oligonucleotide fragment to a 3 ’ oligonucleotide fragment.
  • the prefixes 5’ and 3’ refer to the relative position of each oligonucleotide fragment in the oligonucleotide product after ligation, wherein the 5’ oligonucleotide fragment is located upstream of the 3 ’ oligonucleotide fragment (when the oligonucleotide product is presented in the 5’ to 3’ direction).
  • a “5’ oligonucleotide fragment” typically comprises a 3’ terminal ribonucleotide having a 3 ’-hydroxyl group.
  • a “3’ oligonucleotide fragment” comprises a 5’-phosphate, wherein the 5’ terminal nucleotide is a deoxyribonucleotide or a ribonucleotide.
  • an oligonucleotide fragment may be a 3’ oligonucleotide fragment and a 5’ oligonucleotide fragment (e.g. wherein ligation reactions occur at the 5’ and 3’ ends of the oligonucleotide fragment).
  • said oligonucleotide fragment may provide: (i) the 3’ oligonucleotide fragment in a ligation reaction with a 5’ oligonucleotide fragment; and (ii) the 5 ’ oligonucleotide fragment in a ligation reaction with a 3’ oligonucleotide fragment.
  • oligonucleotide fragment 7 in Figure 1A provides: (i) the 3’ oligonucleotide fragment in a ligation reaction with 5’ oligonucleotide fragment 6 and; (ii) the 5 ’ oligonucleotide fragment in a ligation reaction with 3 ’ oligonucleotide fragment 12 to provide oligonucleotide product 2.
  • a “terminal oligonucleotide fragment” herein refers to a nucleic acid that corresponds to an end (e.g. 5’ or 3’ end) portion of the oligonucleotide product.
  • the 5’ terminal oligonucleotide fragment typically provides a 5’ oligonucleotide fragment for ligation to a 3’ oligonucleotide fragment.
  • the 3’ terminal oligonucleotide fragment typically provides a 3’ oligonucleotide fragment for ligation to a 5 ’ oligonucleotide fragment.
  • the 5’ terminal oligonucleotide is ligated directly to the 3’ terminal oligonucleotide.
  • the 5 ’ terminal oligonucleotide and the 3 ’ terminal oligonucleotide are separated by one or more oligonucleotide fragments.
  • oligonucleotide fragments described herein comprise RNA and DNA.
  • a portion of an oligonucleotide fragment may be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.
  • overhang refers to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of a double -stranded oligonucleotide.
  • this forms a nucleotide overhang, e.g., the unpaired nucleotide(s) form the overhang.
  • An overhang that is complementary to the overhang of a second oligonucleotide fragment may be referred to as a “sticky end”.
  • the oligonucleotide fragments described herein may have one or two sticky ends.
  • Double -stranded nucleic acids comprise two anti-parallel and substantially complementary nucleic acid strands which are referred to as “sense” and “antisense” strands.
  • the “antisense strand” refers to the strand of an RNAi which includes a region that is substantially complementary to a target sequence, e.g. an mRNA sequence.
  • the “sense strand” refers to the strand of an RNAi that includes a region that is substantially complementary to a region of the antisense strand.
  • the sense and antisense strands of an RNAi agent may be referred to as the passenger and guide strands, respectively.
  • Sequences that are “substantially complementary” may be fully complementary or may contain one or more mismatches upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application.
  • Conversion refers to the enzymatic transformation of a substrate to the corresponding product.
  • Percent conversion or “conversion” refers to the percentage of oligonucleotide fragments that is converted to oligonucleotide product within a defined period of time under specified conditions.
  • enzymatic activity or “activity” of a ligase can be expressed as the “percent conversion” of oligonucleotide fragments to oligonucleotide product.
  • £p, £s and £i the extinction coefficient of the product, substrate, and intermediate oligonucleotides respectively.
  • it is possible to resolve at least one substrate, reaction intermediate and product such as well- defined GalNAc-containing oligonucleotides, including GalNAc containing substrate fragments (e.g. oligonucleotide (12) as used in the examples described herein), reaction intermediates (e.g.
  • AU arbitrary units
  • Improved enzyme properties refers to an enzyme property that is better or more desirable for a specific purpose as compared to a reference dsRNA ligase such as a wild-type dsRNA ligase or another engineered dsRNA ligase under the same reaction conditions. Improved enzyme properties are exhibited by engineered dsRNA ligase polypeptides in this disclosure. The engineered dsRNA ligase polypeptides described herein exhibit increased enzyme activity (which can be expressed as a percentage of substrate conversion). Additional enzyme properties that may be improved include, but are not limited to, thermal stability, pH activity characteristics, cofactor requirements, and tolerance to inhibitors (e.g., reaction component, substrate or product inhibition).
  • isolated polypeptide refers to a polypeptide that is substantially separated from other substances with which it is naturally associated, such as proteins, lipids, and polynucleotides.
  • the term comprises polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., in host cells or in vitro synthesis).
  • Engineered dsRNA ligase polypeptides may be present in the cell, in the cell culture medium, or prepared in various forms, such as lysates or isolated preparations.
  • the engineered dsRNA ligase polypeptide may be an isolated polypeptide.
  • Wild-type refers to the form found in nature.
  • a wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism that can be isolated from sources in nature, and which has not been intentionally modified by manual procedures.
  • the polypeptide sequence of the wild-type dsRNA ligase described herein is provided by SEQ ID NO: 302.
  • the wild-type sequence may also comprise a purification tag and may be provided by SEQ ID NO: 2.
  • polynucleotide and “nucleic acid” are used interchangeably herein.
  • protein protein
  • polypeptide and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.).
  • post-translational modification e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.
  • Recombinant or “engineered” when used with reference to, for example, a cell, nucleic acid or polypeptide refers to a material or material corresponding to the native or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic material and/or by manipulation using recombinant techniques.
  • the amino acid may be in either the L- or D-configuration about a-carbon (Ca).
  • “Ala” designates alanine without specifying the configuration about the a-carbon
  • “D-Ala” and “L- Ala” designate D-alanine and L-alanine, respectively.
  • upper case letters designate amino acids in the L-configuration about the a-carbon and lower-case letters designate amino acids in the D-configuration about the a-carbon.
  • A designates L-alanine
  • a designates D-alanine.
  • nucleotides used for the genetically encoding nucleotides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U).
  • the abbreviated nucleotides may be either ribonucleotides or 2 ’-deoxyribonucleotides.
  • the nucleotides may be specified as being either ribonucleotides or 2 ’-deoxyribonucleotides on an individual basis or on an aggregate basis.
  • guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety.
  • a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil.
  • nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of oligonucleotides featured in the present disclosure by a nucleotide containing, for example, inosine.
  • adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form Wobble base pairing with the target mRNA.
  • amino acid difference or “residue difference” refers to the difference in amino acid residues at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in the reference sequence.
  • the positions of amino acid differences are generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence on which the residue differences are based.
  • a residue difference at position X6 as compared to SEQ ID NO: 302 refers to a difference in amino acid residue at the polypeptide position corresponding to position 6 of SEQ ID NO: 302.
  • a residue difference at position X2 as compared to SEQ ID NO: 302 refers to an amino acid substitution to any residue other than serine at the position of the polypeptide corresponding to position 6 of SEQ ID NO: 302.
  • the specific amino acid residue difference at the position may be indicated as “XnY” or “Xn is Y”, wherein “Xn” specifies the corresponding position in the reference sequence as described above, and “Y” is the single letter identifier of the residue present at that position in the engineered polypeptide.
  • Specific amino acid differences may also be denoted by the conventional notation "AnY”, where A is a single letter identifier of the residue in the reference sequence, "n” is the number of residue position in the reference sequence, and “Y” is the single letter identifier of the residue present at that position in the engineered polypeptide.
  • an engineered polypeptide of this disclosure may comprise one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of specific positions at which residue differences are present relative to a reference sequence.
  • more than one amino acid residue can be used in a specific residue position of an engineered polypeptide, the various amino acid residues can be listed as alternatives, e.g. “X19 is Q or D”.
  • Deletion of an amino acid may be represented by e.g. “an amino acid sequence comprising Xn-” indicates that the amino acid sequence contains a deletion at the position corresponding to “Xn” in the reference sequence. “Deletion” refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide.
  • Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids of the enzyme, or up to 20% of the total number of amino acids making up the reference enzyme while retaining the enzymatic activity of the engineered dsRNA ligase and/or retaining the improved properties of the engineered dsRNA ligase. Deletion may involve the internal portion and/or the terminal portion of the polypeptide. In various embodiments, deletions may include a contiguous segment or may be discontinuous.
  • corresponding to refers to the numbering of the residues of a specified reference when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • residue number or residue position of a given sequence is designated with respect to the reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • a given amino acid sequence such as an engineered dsRNA ligase can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although there are gaps, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.
  • Reference sequence refers to a defined sequence that is used as a basis for sequence comparison.
  • the reference sequence may be a subset of a larger sequence, for example, a full-length gene or a fragment of a polypeptide sequence.
  • a “reference sequence” is a wild-type sequence.
  • a “reference sequence” is an engineered or altered sequences.
  • a sequence having a defined number of contiguous nucleotides or amino acids may be aligned with a nucleic acid or peptide sequence (having the same number of contiguous nucleotides or amino acids) from the corresponding portion of a nucleic acid or peptide sequence disclosed herein.
  • the percentage sequence identity can be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences, or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the sequence and multiplying the result by 100 to yield the percentage of sequence identity.
  • Those skilled in the art will appreciate that there are many established algorithms available to align two sequences. The optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, by the Homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.
  • HSPs high scoring sequence pairs
  • T some positive-valued threshold scores
  • the cumulative scores are calculated using the parameters M (reward score for matched pair of residues; always> 0) and N (penalty score for mismatched residues; always ⁇ 0).
  • M forward score for matched pair of residues; always> 0
  • N penalty score for mismatched residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quality X from its maximum achieved value; the cumulative score goes 0 or below, due to the accumulation of one or more negative -scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults the word length (W) of 3, the expected value (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915).
  • Exemplary determination of sequence alignments and %sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using the default parameters provided.
  • an engineered dsRNA ligase possesses dsRNA ligase activity.
  • Suitable reaction conditions refer to those conditions (e.g., enzyme loading, substrate loading, temperature, pH, etc.) in the reaction system, under which the substrate is converted to the desired product. Suitable reaction conditions can be readily identified by the person skilled in the art. Exemplary “suitable reaction conditions” are provided in the present disclosure and illustrated by examples. Engineered dsRNA ligase polypeptides
  • the disclosure provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
  • the disclosure provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668.
  • the disclosure provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
  • the disclosure also provides an engineered dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,
  • engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the disclosure also provides an engineered dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the disclosure also provides an engineered dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,
  • engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • dsRNA double-stranded RNA
  • ligase polypeptide which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
  • a polypeptide having dsRNA ligase activity which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid
  • the disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • dsRNA double-stranded RNA
  • ligase polypeptide which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
  • a polypeptide having dsRNA ligase activity which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600, optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 636-668, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 636-668.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600 or 636-668, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600 or 636-668.
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 exhibit higher activity than that of SEQ ID NO: 302, as shown in the Examples.
  • the dsRNA ligase polypeptides used in the Examples (represented by even numbered sequence identifiers of SEQ ID NOs: 4 to 300 and 602 to 634, respectively) comprise an even numbered sequence identifier of SEQ ID NOs: 304 to 600 and 636 to 668 and an N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 4 comprises: (i) the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 304.
  • dsRNA ligase polypeptides represented by even numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 do not comprise the N-terminal purification tag represented by SEQ ID NO: 669.
  • the wild-type dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 302 (also accessible under UniProt accession number Q7Y4V8).
  • SEQ ID NO: 2 comprises: (i) the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 302. It will be readily understood that SEQ ID NOs: 2 and 302 both comprise the wild-type dsRNA ligase polypeptide sequence and so both sequences may be referred to herein as the wild-type sequence.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590 and 592. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592 and 666.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 188, 226, 278, 288, 290 and 292. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 188, 226, 278, 288, 290, 292 and 632.
  • SEQ ID NOs: 70, 188, 226, 278, 288, 290, 292 and 632 comprise: (i) an N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) an amino acid sequence provided by SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592 and 666, respectively.
  • the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the engineered dsRNA ligase polypeptide polypeptide comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 588.
  • the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666.
  • the disclosure also provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide produces at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions.
  • the ligation reaction conditions are as described herein.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 302, optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 302.
  • the ligation reaction conditions include about 1 pM to about 10 mM oligonucleotide fragment, a source of ATP, about 5 mM to about 100 mM divalent cation, and about 0.5 g/L to about 10 g/L engineered dsRNA ligase polypeptide, pH of about 4.0 to about 8.0, and temperature of about 10 °C to about 50 °C.
  • the source of ATP is a stoichiometric concentration of ATP or a stoichiometric excess of ATP.
  • the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196,
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, XI 14, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163,
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the following amino acid residues:
  • X6 is G or E;
  • X7 is Q;
  • X15 is R, D or E;
  • X19 is Q or D;
  • X29 is N or L;
  • X36 is V;
  • X39 is A;
  • X44 is V;
  • X45 is V;
  • X46 is Y;
  • X47 is E;
  • X49 is G;
  • X51 is L;
  • X53 is Y;
  • X56 is R or A;
  • X57 is S;
  • X60 is T, G or P;
  • X63 is S, Q or G;
  • X64 is R, T, Q, F
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11) amino acid residues selected from: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12) amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12) of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more or three or more) amino acid residues selected from: X36, X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more or three or more) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X36, X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more) amino acid residues selected from: X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more) of the following amino acid residues: X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more or three or more) amino acid residues selected from: X39, X218, X221, and X255; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more orthree or more) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X39, X218, X221, and X255; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, or seven or more) amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more) amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g.
  • X15 is D or E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X15, X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X15 is D; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X15 is E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X 53 is Y; XI 85 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • the engineered dsRNA ligase polypeptide comprises a purification tag.
  • Purification tags are typically appended to polypeptides so that they can be purified from their crude biological source using an affinity technique.
  • the purification tag comprises a poly-histidine tag. Poly-histidine tags bind to matrices bearing immobilized metal ions and can be used to purify polypeptides by affinity chromatography.
  • the purification tag further comprises a protease recognition site for removal of the purification tag.
  • the protease recognition site comprises a Tobacco Etch Virus (TEV) protease recognition sequence.
  • the purification tag comprises the amino acid sequence MHHHHHHENLYFQS (SEQ ID NO: 669).
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.
  • the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
  • 294, 296, 298, 300 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628,
  • the disclosure also provides a polypeptide immobilized on a solid support material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide disclosed herein.
  • Immobilization of a polypeptide by physical absorption typically involves the polypeptide being physically adsorbed or attached onto a solid support material. Adsorption can occur through weak non-specific forces such as van der Waals, hydrophobic interactions and hydrogen bonds. Physical adsorption may be achieved by soaking the support material in a solution of the polypeptide and incubating to allow time for physical adsorption to occur. Immobilization of a polypeptide by chemical bonding typically involves the attachment of the polypeptide to the support material via a covalent bond.
  • the dsRNA ligase polypeptide is immobilized via a spacer positioned between the dsRNA ligase polypeptide and the solid material.
  • the spacer is a peptide (e.g. a peptide comprising 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 75 or more, or 100 or more amino acids).
  • the engineered dsRNA ligase polypeptide is immobilized using affinity immobilization. In some embodiments, the engineered dsRNA ligase polypeptide is immobilized using metal affinity immobilization, e.g. by contacting His- tagged engineered dsRNA ligase polypeptide with immobilized metal such as nickel, zinc, cobalt, or copper.
  • the solid support material comprises a membrane, resin, solid carrier, or other solid phase material.
  • a solid support material can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polymethacrylate, and polyacrylamide, as well as co-polymers and grafts thereof.
  • a solid support material can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum.
  • CPG controlled pore glass
  • the configuration of a solid support material can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non -planar.
  • Solid support materials can be porous or non-porous and can have swelling or non-swelling characteristics.
  • a solid support material can be configured in the form of a well, depression, or other container, vessel, feature, or location.
  • Solid support materials useful for immobilizing the dsRNA ligase polypeptide for carrying out a ligase reaction include but are not limited to beads or resins such as polymethacrylate, e.g., polymethacrylates with epoxy functional groups, polymethacrylates with amino epoxy functional groups, polymethacrylates, styrene/DVB copolymer or polymethacrylates with octadecyl functional groups.
  • Exemplary solid supports include, but are not limited to, chitosan beads, Eupergit C, IB-150, IB-350, IB-C435, IB-A369, IB-A161, IB-A171, IBS500, IB-S861, SEPABEADS (Mitsubishi), e.g., Sepabeads EC-EP, Sepabeads EC-HFA, Sepabeads EC-HG, Sepabeads EC-BU, Sepabeads EC-OD, Sepabeads EC-CM, Sepabeads EC-IDA, Sepabeads EC-EA, Sepabeads EC-HA, Sepabeads EC-QA, Sepabeads EXE, Sepabeads EXA, Dilbeads-TA, Amberzyme Oxirane, Amberlite XAD-7HP, Amberlite FPA98C1, Amberlite IRA
  • this disclosure provides polynucleotides encoding engineered polypeptides having dsRNA ligase activity described herein.
  • the polynucleotides can be linked to one or more heterologous regulatory sequences that control gene expression to produce recombinant polynucleotides that are capable of expressing the engineered polypeptides.
  • Expression constructs comprising a heterologous polynucleotide encoding an engineered dsRNA ligase may be introduced into a suitable host cell to express the corresponding engineered dsRNA ligase polypeptide.
  • this disclosure specifically contemplates each and every possible alteration of a polynucleotide that can be made by selecting a combination based on possible codon selections, for any of the polypeptides disclosed herein, comprising those amino acid sequences of exemplary engineered polypeptides listed in Examples 7 to 12, any of the polypeptides disclosed as even sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668, and any of the polypeptides disclosed as even sequence identifiers of SEQ ID NOs: 4 to 300 and 602 to 634.
  • the codons are preferably selected to accommodate the host cell in which the recombinant protein is produced.
  • codons preferred for bacteria are used to express genes in bacteria; codons preferred for yeast are used to express genes in yeast; and codons preferred for mammals are used for gene expression in mammalian cells.
  • the disclosure provides a polynucleotide encoding an engineered dsRNA ligase polypeptide described above.
  • the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a reference sequence that is an even numbered sequence identifier of SEQ ID NOs: 304 to 600 or 636 to 668, wherein the polypeptide has dsRNA ligase activity and exhibits higher enzyme activity than a polypeptide comprising the amino acid of SEQ ID NO: 2 and/or 302.
  • the polynucleotide encodes an engineered dsRNA ligase polypeptide described herein and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference polynucleotide selected from the sequences having an odd numbered sequence identifier of SEQ ID NOs: 303 to 599 or 635 to 667, wherein: (i) the polynucleotide does not comprise SEQ ID NO: 301; and (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of
  • the polynucleotide encodes an engineered dsRNA ligase polypeptide described herein and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference polynucleotide selected from the sequences having an odd numbered sequence identifier of SEQ ID NOs: 3 to 299 or 601 to 633, wherein: (i) the polynucleotide does not comprise SEQ ID NO: 1; and (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ
  • polynucleotides having an odd numbered sequence identifier of SEQ ID NOs: 3 to 299 or 601 to 633 encode an engineered dsRNA ligase polypeptide comprising an N-terminal purification tag (SEQ ID NO: 669).
  • the isolated polynucleotides encoding engineered dsRNA ligase polypeptides can be manipulated to enable the expression of the engineered polypeptides in a variety of ways, which may comprise further modification of the sequences by codon optimization to improve expression, insertion into suitable expression elements with or without additional control sequences, and transformation into a host cell suitable for expression and production of the engineered polypeptides.
  • manipulation of the isolated polynucleotide prior to insertion of the isolated polynucleotide into the vector may be desirable or necessary.
  • Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided below: Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. Eds., Greene Pub. Associates, 1998, 2010 Year update.
  • the disclosure also provides an expression vector comprising the polynucleotide described herein.
  • the vector is selected from a plasmid, a cosmid, a bacteriophage, or a viral vector.
  • Recombinant expression vectors typically comprise one or more expression regulatory regions, such as promoters and terminators, origin of replication and the like.
  • Polynucleotides encoding an engineered dsRNA ligase polypeptide described herein can be expressed by inserting the polynucleotide or the nucleic acid construct comprising the polynucleotide sequence into an appropriate expression vector.
  • the coding sequence is located in the vector such that the coding sequence is linked to a suitable control sequence for expression.
  • the recombinant expression vector can be any vector (e.g. , a plasmid or virus) that can be conveniently used in recombinant DNA procedures and can result in the expression of a polynucleotide sequence.
  • the choice of vector will generally depend on the compatibility of the vector with the host cell to be introduced into.
  • the vector can be linear or closed circular plasmid.
  • the expression vector may be an autonomously replicating vector, i. e. , a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome.
  • the vector may contain any tools for ensuring self-copying.
  • the vector may be a vector that, when introduced into a host cell, integrates into the genome and replicates with the chromosome into which it is integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together comprise the total DNA to be introduced into the genome of the host cell may be used.
  • An exemplary expression vector can be prepared by inserting a polynucleotide encoding an engineered dsRNA ligase polypeptide to plasmid pACYC-Duet-1 (Novagen), pBR322 Vector (New England Biolabs), pUC19 Vector (New England Biolabs) or pET T7 Expression Vectors (Novagen).
  • the disclosure also provides a host cell capable of expressing an engineered dsRNA ligase polypeptide described herein.
  • the host cell comprises the nucleic acid molecule described herein, or the vector described herein.
  • the host cell is Escherichia coli.
  • the polynucleotide encoding the polypeptide is linked to one or more control sequences for expression of polypeptides in the host cell.
  • Host cells for expression of polypeptides encoded by the expression vectors of the present disclosure are well known in the art, including, but not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium,' fungal cells (e.g., Saccharomyces cerevisiae or Pichia pastoris).' insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells.
  • An exemplary host cell is E. coli BL21 (DE3).
  • the host cell may be wild-type or may be engineered through genomic editing. Suitable media and growth conditions for the above host cells are well known in the art.
  • Polynucleotides or vectors used to express polypeptides can be introduced into cells by a variety of methods known in the art. Techniques comprise, among others, electroporation, bio-particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Different methods of introducing polynucleotides into cells are known to those skilled in the art.
  • the host cell may be used to express and isolate the polypeptide described herein.
  • Engineered dsRNA ligase can be obtained by subjecting a polynucleotide encoding an dsRNA ligase to mutagenesis and/or directed evolution.
  • An exemplary directional evolution technique can be found in "Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing” (2009 John Wiley &Sons Asia (Pte) Ltd. ISBN: 978-0- 470-82314-9).
  • the encoding polynucleotide may be prepared by standard solid-phase methods according to known synthetic methods.
  • fragments of up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence.
  • the polynucleotides and oligonucleotides of the present disclosure can be prepared by chemical synthesis using, for example, the classic phosphoramidite methods described by Beaucage et al., 1981, Tet Lett 22: 1859-69, or Matthes et al. People, 1984, EMBO J.
  • oligonucleotides are synthesized, purified, annealed, ligated, and cloned into a suitable vector, for example, in an automated DNA synthesizer.
  • a suitable vector for example, in an automated DNA synthesizer.
  • essentially any nucleic acid is available from any of a variety of commercial sources.
  • the disclosure provides a method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing a host cell described herein and obtaining an engineered dsRNA ligase polypeptide from the culture.
  • the process of preparing a polypeptide further comprises isolating the polypeptide.
  • Engineered polypeptides may be expressed in suitable cells and isolated (or recovered) from the host cell and/or culture medium using any one or more of the well-known techniques for protein purification, the techniques for protein purification include, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifiigation and chromatography.
  • the invention also provides an engineered dsRNA ligase catalyst obtainable by culturing a host cell described herein, or from the method of preparing an engineered dsRNA ligase polypeptide described herein, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cells, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cells, an extract thereof, or isolated product of the extract.
  • the disclosure provides a method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting: (i) two or more oligonucleotide fragments; (ii) an engineered dsRNA ligase polypeptide disclosed herein; (iii) a source of ATP; and (iv) a divalent cation; to obtain an oligonucleotide.
  • oligonucleotides by ligating two or more oligonucleotide fragments.
  • the produced oligonucleotides are nucleic acids which typically comprise up to 100 nucleotides. It will be understood that oligonucleotides described herein comprise RNA. It will also be understood that oligonucleotides described herein comprise a double-stranded region.
  • oligonucleotide fragment refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide product. Each oligonucleotide fragment corresponds to a portion of the oligonucleotide product.
  • the oligonucleotide is a therapeutic oligonucleotide.
  • the therapeutic oligonucleotide is a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO).
  • the oligonucleotide is an aptamer.
  • the oligonucleotide comprises an overhang. In some embodiments, the oligonucleotide comprises a 3’ overhang. In some embodiments, the oligonucleotide comprises a 5’ overhang. In some embodiments, the overhang comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. In some embodiments, the oligonucleotide comprises a blunt end. In some embodiments, the oligonucleotide comprises two blunt ends.
  • the oligonucleotide is up to 20 nucleotides in length. In some embodiments, the oligonucleotide is up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, or up to 100 nucleotides in length. In some embodiments, the oligonucleotide is up to 60 nucleotides in length.
  • the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or 100 nucleotides in length.
  • the oligonucleotide is 10-100 nucleotides in length. In some embodiments, the oligonucleotide is 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-25 nucleotides in length. In some embodiments, the oligonucleotide is 15-25 nucleotides in length.
  • the two or more oligonucleotide fragments comprise one or more 3’ oligonucleotide fragments and one or more 5 ’ oligonucleotide fragments, wherein each of the one or more 3’ oligonucleotide fragments comprise a 5 ’-phosphate group and each of the one or more 5’ oligonucleotide fragments typically comprise a 3’ terminal ribonucleotide having a 3 ’-hydroxyl group.
  • one or more of the oligonucleotide fragments comprises one or more mismatches. In some embodiments, one or more of the oligonucleotide fragments comprise an overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 3’ overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 5’ overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 3’ overhang and a 5’ overhang. In some embodiments, the overhang comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides.
  • the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having an overhang that is complementary to the overhang of a second oligonucleotide fragment. In some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having a 3 ’ overhang and a 5 ’ overhang, wherein the 3’ overhang is complementary to the 5’ overhang of a second oligonucleotide fragment and the 5’ overhang is complementary to the 3’ overhang of a third oligonucleotide.
  • one or more of the oligonucleotide fragments comprise a blunt end. In some embodiments, one or more of the oligonucleotide fragments comprise a 3’ overhang and a 5’ blunt end. In some embodiments, one or more of the oligonucleotide fragments comprise a 5’ overhang and a 3’ blunt end. In some embodiments, the 5’ terminal oligonucleotide fragment comprises a 3’ overhang and a 5’ blunt end. In some embodiments, the 3’ terminal oligonucleotide fragment comprise a 5’ overhang and a 3’ blunt end.
  • two or more of the oligonucleotide fragments comprise two or more RNA oligonucleotide fragments. In some embodiments, the two or more RNA oligonucleotide fragments comprise double-stranded RNA (dsRNA) oligonucleotide fragments.
  • dsRNA double-stranded RNA
  • one or more of the oligonucleotide fragments comprise DNA and RNA.
  • a portion of the oligonucleotide fragment may be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.
  • one or more of the oligonucleotide fragments comprise one or two strands which are RNA, or a mixture of RNA, DNA, LNA, Morpholino, UNA (unlocked nucleic acid), TNA (threose nucleic acid), GNA (glycol nucleic acid), and/or FANA (Fluoroarabino nucleic acid), modified RNA, etc.
  • one or both strand(s) could be, for example, RNA except that one or more nucleotide(s) is replaced by DNA, LNA, Morpholino, UNA, TNA, GNA, and/or FANA, and/or modified RNA (e.g., any modified RNA disclosed herein or known in the art, such as 2’ modified RNA, including but not limited to 2’-F, 2’-0Me, 2’-0-M0E RNA, etc.).
  • modified RNA e.g., any modified RNA disclosed herein or known in the art, such as 2’ modified RNA, including but not limited to 2’-F, 2’-0Me, 2’-0-M0E RNA, etc.
  • the two or more oligonucleotide fragments are the same length. In some embodiments, the two or more oligonucleotide fragments are different lengths. In some embodiments, each of the two or more oligonucleotide fragments are 3-20 nucleotides in length. In some embodiments, each of the two or more oligonucleotide fragments are 4-16 nucleotides in length. In some embodiments, each of the two or more oligonucleotide fragments are 4-16, 4-15, 5-15, 6-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 5-9, or 6-9 nucleotides in length.
  • each of the two or more oligonucleotide fragments are at least 3 nucleotides in length. In some embodiments, each of the two or more oligonucleotide fragments are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
  • the two or more oligonucleotide fragments comprise 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more oligonucleotide fragments.
  • one or more ligation reactions are required to generate the oligonucleotide product. In some embodiments, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more ligation reactions are required to generate the oligonucleotide product.
  • one or more of the oligonucleotide fragments and/or the oligonucleotide comprises a chemical modification. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one modified backbone modification. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one modified nucleotide modification. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one sugar modification (e.g. at the 2’-position or 4’-position).
  • one or more of the oligonucleotide fragments and/or the oligonucleotide comprises: (i) at least one modified backbone modification; (ii) and at least one modified nucleotide modification; and/or (iii) at least one sugar modification.
  • one or more of the oligonucleotide fragments and/or the oligonucleotide comprise a modification selected from the group consisting of: 2'-O-methyl (2’-OMe), 2'-flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'- O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'- O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g.
  • mesyl phosphoramidate 2',3'-seco nucleotide mimic
  • 2'-F-arabino nucleotide abasic nucleotide
  • 2'-amino modified nucleotide 2'-alkyl-modified nucleotide
  • morpholino nucleotide vinylpho sphonate (e.g. 5’ vinylphosphonate)
  • cyclopropyl phosphonate deoxyribonucleotide 2',3'-seco nucleotide mimic
  • 2'-F-arabino nucleotide abasic nucleotide
  • 2'-amino modified nucleotide 2'-alkyl-modified nucleotide
  • morpholino nucleotide e.g. 5’ vinylphosphonate
  • cyclopropyl phosphonate deoxyribonucleotide cyclopropyl phosphonate deoxyribonucleotide
  • one or more of the oligonucleotide fragments and/or the oligonucleotide comprises a 2'-modification selected from the group consisting of: 2’-0Me, 2’-F, and 2'-deoxy.
  • one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one phosphorothioate or methylphosphonate intemucleotide linkage. In some embodiments, the oligonucleotide comprises at least one chiral phosphorothioate linkage.
  • one or more of the oligonucleotide fragments and/or the oligonucleotide is conjugated to at least one ligand.
  • the ligand may be conjugated to the sense strand, antisense strand or both strands, in any configuration e.g. at the 3 ’-end, 5 ’-end, non-end or a combination.
  • the ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives.
  • GalNAc is an amino sugar derivative of galactose which may be used as a targeting ligand in oligonucleotides intended for targeting to the liver, where it binds to the asialoglycoprotein receptors on hepatocytes.
  • the ligand comprises one or more GalNAc derivatives conjugated through a bivalent or trivalent branched carrier.
  • the ligand is a peptide or a peptidomimetic.
  • the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3’ end of the sense strand. In some embodiments, the ligand is conjugated to the 5’ end of the sense strand. In some embodiments, the ligand is conjugated to a non-end of the sense strand.
  • the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the 3’ end of the antisense strand. In some embodiments, the ligand is conjugated to a non-end of the antisense strand.
  • the oligonucleotide is an RNAi agent comprising at least one 2’-modified nucleotide selected from a group consisting of 2’-0Me, 2’-F, 2'-deoxy, 2'-deoxy- 2’-fluoro, and 2'-0-M0E.
  • the oligonucleotide is an RNAi agent wherein the sense strand is conjugated to one or more GalNAc ligand(s).
  • one or more of the oligonucleotide fragments comprises at least one 2’- modified nucleotide selected from a group consisting of 2’-0Me, 2’-F, 2'-deoxy, 2'-deoxy-2’- fluoro, and 2'-0-M0E.
  • one or more of the oligonucleotide fragments is a dsRNA wherein the sense strand is conjugated to one or more GalNAc ligand(s).
  • the method is performed with an oligonucleotide fragment concentration of at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM. In some embodiments, the method is performed with at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM of each oligonucleotide fragment. In some embodiments, the method is performed with equimolar amounts of each of the two or more oligonucleotide fragments.
  • the method produces at least 15 g of oligonucleotide product per litre of reaction mixture. In some embodiments, the method produces at least 16 g, at least 17 g, at least 18 g, at least 19 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90, or at least 100 g of oligonucleotide product per litre of reaction mixture.
  • the method is performed using an engineered dsRNA ligase as described herein.
  • the method is performed using about 1 g/L engineered dsRNA ligase polypeptide, optionally 1.1 g/L, 1.15 g/L, 1.2 g/L, 1.25 g/L, 1.3 g/L, 1.35 g/L, 1.4 g/L, 1.45 g/L, 1.5 g/L, 1.55 g/L, 1.6 g/L, 1.65 g/L, 1.7 g/L, 1.75 g/L, 1.8 g/L, 1.85 g/L, 1.9 g/L, 1.95 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.25 g/L, 3.5 g/L, 3.75 g/L, 4 g
  • dsRNA ligase The enzymatic activity of dsRNA ligase requires ATP as a cofactor. One molecule of ATP is converted to AMP per ligation reaction. The catalytic mechanism of dsRNA ligase and the role of ATP in nucleic acid ligation reactions are described above.
  • the source of ATP is ATP.
  • the method is performed using a stoichiometric concentration of ATP.
  • the method is performed using a stoichiometric excess of ATP. The skilled person can readily determine the stoichiometric concentration of ATP required for a given ligation based on the concentration of the oligonucleotide fragments and the number of ligation reactions required to produce the oligonucleotide product.
  • the method is performed using an ATP and/or AMP concentration of about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28mM or about 30mM.
  • an ATP and/or AMP concentration of about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM
  • the source of ATP is an ATP regeneration system.
  • the ATP regeneration system comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
  • PPK polyphosphate kinase
  • AMP AMP and/or ATP.
  • the ATP regeneration system described herein comprises PPK and polyphosphate. PPK generates ATP from AMP using polyphosphate as a phosphate donor. ATP that is converted to AMP during the ligation reaction can be regenerated to ATP by PPK and used as a cofactor in a subsequent ligation reaction. This cycling of ATP obviates the need for high ATP concentration in the starting reaction. Instead, the reaction can be performed using sub- stoichiometric concentrations of ATP, and/or using the cheaper alternative, AMP.
  • Polyphosphate kinases or “PPKs” are a family of enzymes which catalyze the formation of ATP from AMP and polyphosphate.
  • the PPK is PPK12.
  • the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 670, which is the amino acid sequence of PPK12.
  • the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 670.
  • the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 671, which is the amino acid sequence of an optimized PPK12. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 671.
  • the PPK is Acinetobacter johnsonii polyphosphate: AMP phosphotransferase (AjPAP) (UniProt ID: Q83XD3).
  • the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 672, which is the amino acid sequence of AjPAP.
  • the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 672.
  • the PPK is used in the form of whole cell, crude extract (e.g. cell free lyophilized extract or cell lysates), isolated polypeptide, or purified polypeptide.
  • the PPK polypeptide is used in an immobilized form as described herein, such as immobilized on a resin.
  • the method is performed using about 1 g/L PPK, optionally 1.1 g/L, 1.15 g/L, 1.2 g/L, 1.25 g/L, 1.3 g/L, 1.35 g/L, 1.4 g/L, 1.45 g/L, 1.5 g/L, 1.55 g/L, 1.6 g/L, 1.65 g/L, 1.7 g/L, 1.75 g/L, 1.8 g/L, 1.85 g/L, 1.9 g/L, 1.95 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.25 g/L, 3.5 g/L, 3.75 g/L, 4 g/L, 4.5 g/L or 5
  • the polyphosphate is a polyphosphate salt.
  • the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
  • the method is performed using a stoichiometric excess of polyphosphate. In some embodiments, the method is performed using a polyphosphate concentration of at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.
  • the method is performed in the presence of PPK and polyphosphate
  • the method is performed in the presence of AMP.
  • the method is performed using a sub-stoichiometric concentration of ATP and/or AMP.
  • the enzymatic activity of dsRNA ligases requires the presence of a divalent cation.
  • the enzymatic activity of PPKs requires the presence of a divalent cation.
  • the divalent cation comprises Mg 2+ and/or Mn 2+ .
  • the method is performed with a divalent cation concentration of 5-100 mM, 10-100 mM, 15-100 mM, 20-100 mM, 30-100 mM, 5-90 mM, 5-80 mM, 5-70 mM, 5-60 mM, 5-50 mM, or 30-50 mM.
  • the method is performed with a divalent cation concentration of at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.
  • the method further comprises purifying the oligonucleotide product from the reaction mixture.
  • the oligonucleotide product is at least 80% pure, optionally wherein the oligonucleotide product is at least 85%, at least 90%, at least 95% pure, optionally wherein the oligonucleotide product is at least 98% pure, optionally wherein the oligonucleotide product is at least 99% pure, optionally wherein the oligonucleotide product is at least 99.5% pure, optionally wherein the oligonucleotide product is at least 99.9% pure.
  • oligonucleotide product that is pure does not contain oligonucleotide fragments, intermediate ligation products, or side products arising from nonspecific ligation.
  • the oligonucleotide product may be purified or isolated using any method known in the art, for example using gel extractions or using cellulose-based matrices.
  • the disclosure also provides an oligonucleotide produced by a method described herein.
  • the oligonucleotide may be in any suitable buffer solution.
  • the buffer solution is selected from Tris buffer (e.g. Tris-HCl), phosphate buffer, HEPES, MOPS (3-(A-morpholino)propancsulfonic acid), and triethanolamine (TEOA) buffer.
  • the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution further comprises an agent for controlling the osmolarity of the solution, such that the osmolarity is kept at a desired value, e.g., at the physiologic values of the human plasma.
  • Solutes which can be added to the buffer solution to control the osmolarity include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts.
  • the agent for controlling the osmolarity of the solution is a salt.
  • the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride.
  • the present disclosure contemplates a range of suitable reaction conditions that may be used in the methods described herein, including but not limited to pH, temperature, buffers, substrate loadings, enzyme loading, cofactor loading, pressure, and reaction time. Additional suitable reaction conditions for ligation reactions described herein can be readily optimized by routine experimentation, e.g. performing the method described herein under experimental reaction conditions of varying reagent concentration, pH, temperature, and detecting the rate of oligonucleotide product formation.
  • the reaction conditions may include a suitable pH.
  • the desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer, or a combination of buffer and added acid or base.
  • the pH of the reaction mixture can be controlled before and/or during the reaction.
  • suitable reaction conditions include a solution pH of about 4 to about 8, a pH of about 5 to about 8, a pH of about 6 to about 8, or a pH of about 7 to about 8.
  • the reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.
  • suitable temperatures can be used for the reaction conditions, taking into consideration of, for example, the increase in reaction rate at higher temperatures, the activity of the enzyme for sufficient duration of the reaction.
  • suitable reaction conditions include a temperature of about 10°C to about 60°C, about 10°C to about 50°C, about 25 °C to about 50°C, about 25°C to about 40°C, about 25°C to about 30°C, or about 10°C to about 30°C.
  • suitable reaction temperatures include a temperature of about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C.
  • the temperature during the enzymatic reaction can be maintained at a certain temperature throughout the reaction. In some embodiments, the temperature during the enzymatic reaction may be adjusted over a temperature profile during the course of the reaction.
  • the reaction may be performed in any suitable buffer solution.
  • the buffer solution is selected from Tris buffer (e.g. Tris-HCl), phosphate buffer, HEPES, MOPS (3-(A-morpholino)propanesulfonic acid), and triethanolamine (TEOA) buffer.
  • the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof.
  • the buffer solution is phosphate buffered saline (PBS).
  • the reaction mixture further comprises a reducing agent, optionally DTT (Dithiothreitol).
  • a reducing agent optionally DTT (Dithiothreitol).
  • the engineered dsRNA ligase polypeptide may be added to the reaction mixture in different formulation forms, as frozen or lyophilized whole cells (FWC or LWC) transformed with the gene encoding the engineered dsRNA ligase polypeptide and/or as cell lysate or lyophilized cell lysate of such cells, so called shake flask powder (SFP), where the cell debris was removed and/or further purified as fermentation powder (FP).
  • FWC or LWC frozen or lyophilized whole cells transformed with the gene encoding the engineered dsRNA ligase polypeptide and/or as cell lysate or lyophilized cell lysate of such cells, so called shake flask powder (SFP), where the cell debris was removed and/or further purified as fermentation powder (FP).
  • SFP shake flask powder
  • Whole cells transformed with gene(s) encoding the engineered dsRNA ligase polypeptide or cell extracts, lysates thereof, and isolated enzymes can be used in a wide variety of different forms, including solids (e.g., lyophilized, spray dried, or the like) or semisolid (e.g., a crude paste).
  • the cell extract or cell lysate may be partially purified by precipitation (e.g. , ammonium sulfate, polyethyleneimine, heat treatment or the like), followed by desalting procedures (e.g., ultrafiltration, dialysis, and the like) prior to lyophilization.
  • Any of the enzyme preparations can be immobilized to a solid phase material (such as a resin).
  • a culture medium containing the secreted polypeptide can be used in the process herein.
  • the solid reactants e.g. , enzymes, salts, etc.
  • the reaction can be provided to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray dried, etc.), solutions, emulsions, suspensions, and the like.
  • the reactants can be readily lyophilized or spray-dried using methods and instrumentation known to one skilled in the art.
  • the protein solution can be frozen at -80 °C in small aliquots, and then added to the pre-chilled lyophilization chamber, followed by the application of a vacuum.
  • the order of addition of reactants is not critical.
  • the reactants may be added together to the solvent at the same time or alternatively, some reactants may be added separately, and some may be added together at different time points.
  • the methods of performing a ligation reaction may comprise the further step of isolating the oligonucleotide product of the enzymatic reaction. In particular, this step is typically performed after completion of the enzymatic reaction.
  • the oligonucleotide is in particular typically separated from one or more, in particular essentially all of the other components of the reaction mixture. For example, the oligonucleotide is typically separated from the remaining substrate, side products, and/or enzymes. Isolation of the oligonucleotide may be achieved by means and techniques known in the art, e.g. by separating oligonucleotides based on their size such as by gel electrophoresis and gel extractions or using cellulose-based matrices.
  • the method further comprises purifying the oligonucleotide by ultrafiltration and chromatography. Modifications
  • the oligonucleotide fragment(s) and/or the oligonucleotide comprises a modification, e.g. a chemical modification.
  • oligonucleotide fragment(s) means one or more oligonucleotide fragments. It will be appreciated that modifications which are present in the oligonucleotide fragment(s) are typically present in the oligonucleotide produced from said oligonucleotide fragment(s). In some embodiments, modification(s) are introduced to and/or removed from the oligonucleotide product.
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises a chemical modification. In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one backbone modification. In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one nucleotide modification. In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one sugar modification (e.g. at the 2’-position or 4’-position).
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises: (i) at least one backbone modification; (ii) at least one nucleotide modification; and/or (iii) at least one sugar modification.
  • Modifications include, but are not limited to, end modifications of the terminal oligonucleotide fragments, e.g., 5 ’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, inverted linkages, etc.); base modifications, e.g. , replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications of the terminal oligonucleotide fragments e.g., 5 ’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, inverted linkages, etc.
  • base modifications e.g. , replacement with stabilizing bases, destabilizing bases, or bases that base
  • a terminal oligonucleotide fragment and/or oligonucleotide comprises a cap.
  • the term "cap” and the like include a chemical moiety attached to the end of a double-stranded nucleotide duplex, but is used herein to exclude a chemical moiety that is a nucleotide or nucleoside.
  • a “3’ cap” is attached at the 3’ end of a nucleotide or oligonucleotide and protects the molecule from degradation, e.g., from nucleases, such as those in blood serum or intestinal fluid.
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises one or more mismatches.
  • a mismatch is defined herein as a difference between the base sequence or length when two sequences are maximally aligned and compared.
  • a mismatch is defined as a position wherein the base of one sequence is not complementary to the base of the other sequence.
  • a mismatch is counted, for example, if a position in the first sequence has a particular base (e.g., A), and the corresponding position in the second sequence has a base which is not complementary to said base in the first sequence (e.g., G), when the first and second sequences are aligned antiparallel to each other.
  • a U can be replaced by T (either as RNA or, preferably, DNA, e.g., 2 ’-deoxy-thymidine); the replacement of a U with a T is not a mismatch as used herein, as either U or T can pair with A on the opposite strand.
  • RNA oligonucleotide can thus comprise one or more DNA bases, e.g., T. No mismatch is counted between a DNA portion(s) of an RNAi agent and the corresponding target mRNA if basepairing occurs (e.g., between A, G, C, or T in the DNA portion, and the corresponding U, C, G, or A, respectively in the mRNA).
  • a nucleotide modification in the sugar or phosphate is also not considered a mismatch.
  • one sequence comprises a G
  • the complementary sequence comprises a modified C (e.g., 2 ’-modification) at the same position, no mismatch would be counted.
  • RNAi double-stranded RNAi
  • a strand having a given sequence as an RNA would have zero mismatches from its complement sequence as a PNA; or morpholino; or LNA; or TNA; or GNA; or FANA; or a mix or chimera of RNA and DNA, TNA, GNA, FANA, Morpholino, UNA, LNA, and/or PNA, etc.
  • mismatch No mismatch would occur between a nucleotide which is T, and a nucleotide which is A with a 5’ modification and/or a 2 ’-modification.
  • base replacement base replacement
  • terminal overhangs such as “UU” or “dTdT” are not counted when counting the number of mismatches.
  • a mismatch is defined as a position wherein the base of one sequence does not match the base of the other sequence.
  • dTdT (2'-deoxy-thymidine-5 ’-phosphate and 2'-deoxy-thymidine-5’- phosphate), or in some cases, TT or UU
  • TT or UU can be added as a terminal dinucleotide cap or extension to one or both 3 ’-ends of the oligonucleotide, but this cap or extension is not included in the calculation of the total number of mismatches and is not considered part of the target sequence.
  • the terminal dinucleotide protects the ends from nuclease degradation but does not contribute to target specificity (Elbashir et al. 2001 Nature 411: 494- 498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12: 163- 176).
  • nucleic acid molecules There are several examples in the art describing sugar, base, phosphate and backbone modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base modifications.
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base modifications.
  • Sugar modification of nucleic acid molecules are extensively described in the art.
  • oligonucleotides Additional modifications and conjugations have been described. Soutschek et al. 2004 Nature 432: 173-178 presented conjugation of cholesterol to the 3’-end of the sense strand of an siRNA molecule by means of a pyrrolidine linker, thereby generating a covalent and irreversible conjugate. Chemical modifications (including conjugation with other molecules) of oligonucleotides may also be made to improve the in vivo pharmacokinetic retention time and efficiency.
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises a modified base.
  • the disclosure encompasses an oligonucleotide and oligonucleotide fragments with a substitution of a single nucleotide at a given position with a modified version of the same nucleotide.
  • a nucleotide (A, G, C or U) can be replaced by a modified base selected from 5-fluorouracil, 5 -bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5 -(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2 -thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5 -methylcytosine, N6-adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5- methoxyaminomethyl-2 -thiouracil, beta-D-
  • Additional modified variants include the addition of any other moiety (e.g., a radiolabel or other tag or conjugate) to the oligonucleotide or oligonucleotide fragment; provided that the base sequence is identical, the addition of other moieties produces a “modified variant” (with no mismatches).
  • any other moiety e.g., a radiolabel or other tag or conjugate
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises a modification that causes the oligonucleotide to have increased stability in a biological sample or environment (e.g., cytoplasm, interstitial fluid, blood serum, lung or intestinal lavage).
  • a biological sample or environment e.g., cytoplasm, interstitial fluid, blood serum, lung or intestinal lavage.
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises a modification that promotes cleavage by the RNA-induced silencing complex (z. e. a “RISC cleavage site”).
  • the RISC cleavage site is the site on the target at which cleavage occurs.
  • the antisense strand comprises a RISC cleavage site.
  • the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end.
  • the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13 of the antisense strand.
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises a modified backbone.
  • an unmodified backbone consists of 3’ to 5’ phosphodiester bonds.
  • a modified backbone may comprise non-natural intemucleoside linkages.
  • Oligonucleotides having a modified backbone include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • Oligonucleotide fragments comprising a modified backbone include, but are not limited to, those that do not have a phosphorus atom in the backbone.
  • Modified backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (e.g. 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (e.g.
  • Oligonucleotide fragments comprising a modified backbone that does not include a phosphorus atom therein may have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts.
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises at least one phosphonate linkage, wherein the phosphonate is a modified phosphonate selected from the group consisting of: phosphorothioate (which may be an Rp isomer or an .S'p isomer):
  • methylphosphonate methoxypropylphosphonate : 5 ’ -methylphosphonate : phonate: 5’-phosphorothioate; and peptide nucleic acid:
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises: at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’-modified nucleotide; at least one 5’-uridine-guanine-3’ (5’-ug-3’) dinucleotide, wherein the 5 ’-uridine is a 2’-modified nucleotide; at least one 5’-cytidine-adenine-3’ (5’-ca-3’) dinucleotide, wherein the 5’-cytidine is a 2’-modified nucleotide; or at least one 5’-uridine- uridine-3’ (5’-uu-3’) dinucleotide, wherein the 5’-uridine is a 2’-modified nucleotide.
  • dinucleotide motifs are particularly prone to serum nuclease degradation (e.g. RNase A).
  • Chemical modification at the 2'-position of the first pyrimidine nucleotide in the motif prevents or slows down such cleavage.
  • This modification recipe is also known under the term 'endo light'.
  • the oligonucleotide and/or oligonucleotide fragment(s) comprise a modified nucleobase, wherein the modified nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particular embodiment, the modified nucleobase is difluorotolyl. In some embodiments, wherein the oligonucleotide and/or oligonucleotide fragment(s) is double-stranded, only one of the two strands contains a modified nucleobase. In some embodiments, wherein the oligonucleotide and/or oligonucleotide fragment(s) is double-stranded, both of the strands contain a modified nucleobase.
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises a modified sugar.
  • Sugar modifications typically involve chemical modification of the sugar moiety of RNA or DNA.
  • Sugar modifications include, but are not limited to, one of the following at the 2’-position: OH; F; O-, S-, orN-alkyl; O-, S-, orN-alkenyl; O-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to Cio alkyl or C2 to Cio alkenyl and alkynyl.
  • Exemplary modifications include O[(CH 2 ) n O] mCHs, O(CH 2 ). n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
  • Oligonucleotide fragments for use in the methods described herein may include one of the following at the 2’ position: Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a therapeutic RNA, or a group for improving the pharmacodynamic properties of a therapeutic RNA.
  • the modification comprises a 2 ’-methoxy ethoxy (also known as 2 ’-O-(2 -methoxyethyl) or 2’-O-MOE), 2’- dimethylaminooxyethoxy (also known as 2’-DMAOE), and 2 ’-dimethylaminoethoxy ethoxy (also known in the art as 2’-O-dimethylaminoethoxyethyl or 2’-DMAEOE).
  • 2 ’-methoxy ethoxy also known as 2 ’-O-(2 -methoxyethyl) or 2’-O-MOE
  • 2’- dimethylaminooxyethoxy also known as 2’-DMAOE
  • 2 ’-dimethylaminoethoxy ethoxy also known in the art as 2’-O-dimethylaminoethoxyethyl or 2’-DMAEOE
  • modifications include: 5’-Me-2’-F nucleotides, 5’-Me-2’-Ome nucleotides, 5’- Me -2 ’-deoxynucleotides, 2 ’-alkoxyalkyl; and 2’-NMA (N-methylacetamide).
  • Other modifications include 2’-methoxy (2’-OCH3), 2 ’-aminopropoxy (2’- OCH2CH2CH2NH2) and 2 ’-fluoro (2’-F). Similar modifications can also be made at other positions on an RNA, particularly the 3 ’ position of the sugar on the 3 ’ terminal nucleotide or in 2’-5’ linked dsRNA and the 5’ position of 5’ terminal nucleotide.
  • the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one modified nucleotide.
  • the modification is selected from the group consisting of: 2’-O-methyl (2’-0me), 2’-flouro (2’-F), 2’-deoxy, 2’- deoxy-2’ -fluoro, 2’-O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl (2’-O-AP), 2’-O- dimethylaminoethyl (2’-0-DMA0E), 2’-O-dimethylaminopropyl (2’-0-DMAP), 2 -0- dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’-O-N-methylacetamido (2’-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.
  • one or more of the oligonucleotide fragments comprises a 2 ’-modification selected from the group consisting of: 2’-0me, 2’-F, and 2’- deoxy.
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises one or more 3'-O-methyl nucleotide.
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises a 2'-modification selected from the group consisting of: 2'-O-methyl (2’-0Me), 2'- flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-0-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O- dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-0-DMAE0E), 2'- O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), phosphoramidate (e.g.
  • oligonucleotide and/or oligonucleotide fragment(s) comprises one or more 3'-O-methyl nucleotide.
  • the oligonucleotide and/or oligonucleotide fragment(s) comprises a bridged nucleic acid.
  • the bridged nucleic acid is locked nucleic acid.
  • the bridged nucleic acid is constrained ethyl bridged nucleic acid:
  • all pyrimidines are 2’ O-methyl- modified nucleosides.
  • the sense and/or antisense strand is conjugated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, modified or unmodified nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium- molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.
  • the antisense strand comprises at least one 2’-0Me modified nucleotide. In some embodiments, the antisense strand comprises at least one 2’-F modified nucleotide. In some embodiments, the antisense strand comprises at least one 2’-deoxy modified nucleotide. In some embodiments, the antisense strand comprises at least one 2’- OMe modified nucleotide, at least one 2’-F modified nucleotide, or at least one 2’-deoxy modified nucleotide, or any combination thereof. In some embodiments, the antisense strand comprises alternating 2’-0Me and 2’-F modified nucleotides.
  • the antisense strand comprises at least one 5’ vinylphosphonate. In some embodiments, the antisense strand comprises at least one chiral phosphorothioate linkage. In some embodiments, the antisense strand comprises at least one GNA. In some embodiments, the sense strand comprises at least one 2’-0Me modified nucleotide. In some embodiments, the sense strand comprises at least one 2’-F modified nucleotide. In some embodiments, the sense strand comprises at least one 2’-deoxy modified nucleotide.
  • the sense strand comprises at least one 2’-0Me modified nucleotide, at least one 2’-F modified nucleotide, or at least one 2’ -deoxy modified nucleotide, or any combination thereof. In some embodiments, the sense strand comprises alternating 2’-0Me and 2’-F modified nucleotides. In some embodiments, the antisense strand and the sense strand each comprise at least one 2’-0Me modified nucleotide. In some embodiments, the antisense strand and the sense strand each comprise at least one 2’-F modified nucleotide.
  • the antisense strand and the sense strand each comprise alternating 2’-0Me and 2’-F modified nucleotides.
  • the sense strand comprises at least one 5’ vinylphosphonate.
  • the sense strand comprises at least one chiral phosphorothioate linkage.
  • the sense strand comprises at least one GNA.
  • the sense strand comprises alternating 2’-0Me and 2’-F modified nucleotides over the full length of the sense strand. In some embodiments, the sense strand comprises alternating 2’-0Me and 2’-F modified nucleotides over part of the length of the sense strand e.g. over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides of the sense strand.
  • the antisense strand comprises alternating 2’-OMe and 2’-F modified nucleotides over the full length of the antisense strand. In some embodiments, the antisense strand comprises alternating 2’-OMe and 2’-F modified nucleotides over part of the length of the antisense strand e.g. over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • nucleotides of the antisense strand 17, 18, 19, 20 or more nucleotides of the antisense strand.
  • the sense strand and antisense strand each comprise alternating 2’-OMe and 2’-F modified nucleotides over the full length of the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand comprise alternating 2’-OMe and 2’-F modified nucleotides over part of the length of the sense strand and the antisense strand e.g. over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • nucleotides of the sense strand and the antisense strand 18, 19, 20 or more nucleotides of the sense strand and the antisense strand.
  • one or more of the oligonucleotide fragments is conjugated to at least one ligand.
  • the oligonucleotide product is conjugated to at least one ligand.
  • the ligand may be conjugated to the sense strand, antisense strand or both strands, in any configuration e.g. at the 3 ’-end, 5 ’-end, non-end or a combination.
  • the ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives. In some embodiments, the ligand comprises one or more GalNAc derivatives conjugated through a bivalent or trivalent branched carrier. In some embodiments, the ligand is:
  • the ligand is:
  • the ligand is: In some embodiments, the ligand is:
  • the ligand is:
  • the ligand is:
  • a ligand alters the distribution, targeting or lifetime of the molecule into which it is incorporated.
  • a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
  • Ligands providing enhanced affinity for a selected target are also termed targeting ligands.
  • Some ligands can have endosomolytic properties.
  • the endosomolytic ligands promote the lysis of the endosome and/or transport of the oligonucleotide, or a composition comprising the oligonucleotide, from the endosome to the cytoplasm of the cell.
  • the endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH- dependent membrane activity and fusogenicity. In some embodiments, the endosomolytic ligand assumes its active conformation at endosomal pH.
  • the “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the oligonucleotide, or a composition comprising the oligonucleotide, from the endosome to the cytoplasm of the cell.
  • exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68).
  • the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH.
  • the endosomolytic component may be linear or branched.
  • Ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligonucleotide.
  • Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties.
  • therapeutic modifiers e.g., for enhancing uptake
  • diagnostic compounds or reporter groups e.g., for monitoring distribution
  • cross-linking agents e.g., for monitoring distribution
  • nuclease-resistance conferring moieties lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
  • Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid.
  • the ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer).
  • polyamino acids examples include polylysine (PLL), poly L aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.
  • PLL polylysine
  • poly L aspartic acid poly L-glutamic acid
  • styrene-maleic acid anhydride copolymer examples include poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copoly
  • polyamines examples include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide .
  • Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type.
  • a targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N- acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptidomimetic or an aptamer.
  • ligands include dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelator (e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis- O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(ol),
  • Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
  • Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers.
  • the ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF -KB .
  • the ligand is a lipid or lipid-based molecule.
  • a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA).
  • HSA binding ligand allows for distribution of the conjugate to a target tissue.
  • a lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
  • a lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue.
  • the ligand is a peptide or a peptidomimetic.
  • a peptidomimetic is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • a peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe).
  • the peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide.
  • the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).
  • MTS membrane translocation sequence
  • the peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes.
  • a peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phagedisplay library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991).
  • a “peptide moiety” can range in length from about 5 amino acids to about 50 amino acids.
  • the peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
  • An arginine-glycine-aspartic acid (RGD)-peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002).
  • RGD peptide can facilitate targeting of an oligonucleotide to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001).
  • the RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues.
  • Peptides that target markers enriched in proliferating cells can be used.
  • RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin.
  • the ligand may comprise RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, or synthetic RGD mimics.
  • Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, , or y peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides .
  • Ligands can be coupled to the oligonucleotide fragment(s) and/or oligonucleotide at various places, for example, 3 ’-end, 5 ’-end, and/or at an internal (“non-end”) position.
  • the ligand is attached via an intervening tether, e.g., a carrier described herein.
  • the ligand or tethered ligand may be present on a monomer when the monomer is incorporated into the oligonucleotide fragment(s) and/or oligonucleotide.
  • the ligand may be incorporated via coupling to a “precursor” monomer after the “precursor” monomer has been incorporated into the oligonucleotide fragment and/or the oligonucleotide.
  • a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH2)nNH2 may be incorporated into a growing oligonucleotide fragment.
  • a ligand having an electrophilic group e.g., a pentafluorophenyl ester or aldehyde group
  • a ligand having an electrophilic group can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer’s tether.
  • a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated, e.g., an azide or alkyne terminated tether/linker.
  • a ligand having complementary chemical group e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
  • the ligand is conjugated to nucleobases, sugar moieties, or intemucleosidic linkages of the oligonucleotide fragment(s) and/or oligonucleotide.
  • Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms.
  • the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety.
  • Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position.
  • the 2-, 5-, and 6- positions of a pyrimidine nucleobase can be substituted with a conjugate moiety.
  • Conjugation to sugar moieties of nucleosides can occur at any carbon atom.
  • Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms.
  • the T position can also be attached to a conjugate moiety, such as in an abasic residue.
  • Intemucleosidic linkages can also bear conjugate moieties.
  • phosphorus- containing linkages e.g., phosphodiester, phosphorothioate (e.g.
  • the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom.
  • the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
  • the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3’ end of the sense strand. In some embodiments, the ligand is conjugated to the 5’ end of the sense strand. In some embodiments, the ligand is conjugated to a non-end of the sense strand.
  • the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the 3’ end of the antisense strand. In some embodiments, the ligand is conjugated to a non-end of the antisense strand.
  • the ligand may be attached via a carrier.
  • the carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.”
  • a “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a nucleic acid.
  • a “tethering attachment point” in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety.
  • the moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide.
  • the selected moiety is connected by an intervening tether to the cyclic carrier.
  • the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
  • a functional group e.g., an amino group
  • another chemical entity e.g., a ligand to the constituent ring.
  • the sense and/or antisense strand may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.
  • one or more oligonucleotide fragments comprise the sequence “TT”, “dTdT”, “dTsdT” or “UU” as a single-stranded overhang at the 3’ end, also termed herein a terminal dinucleotide or 3’ terminal dinucleotide.
  • dT is 2'-deoxy-thymidine-5’- phosphate
  • sdT is 2'-deoxy Thymidine 5'-phosphorothioate.
  • Terminal dinucleotide “UU” is UU or 2’-0Me-U 2’-0Me-U, and the terminal TT and the terminal UU can be in the inverted/reverse orientation.
  • the terminal dinucleotide (e.g., UU) is a modified variant of the dithymidine dinucleotide commonly placed as an overhang to protect the ends of siRNAs from nucleases (see, for example, Elbashir et al. 2001 Nature 411: 494-498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12: 163-176).
  • a terminal dinucleotide is known from these references to enhance nuclease resistance but not contribute to target recognition.
  • one or both terminal oligonucleotide fragments comprise a 3 ’ end cap instead of or in addition to a terminal dinucleotide to stabilize the end from nuclease degradation provided that the 3’ end cap is able to both stabilize the oligonucleotide (e.g., against nucleases) and not interfere excessively with its desired activity.
  • the sense and/or antisense strand may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.
  • Embodiment 1 An engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,
  • dsRNA double-stranded RNA
  • engineered dsRNA ligase polypeptide (a) has dsRNA ligase activity;
  • Embodiment 2 An engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,
  • dsRNA double-stranded RNA
  • Embodiment 3 The engineered dsRNA ligase polypeptide of Embodiment 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592.
  • Embodiment 4 The engineered dsRNA ligase polypeptide of Embodiment 2, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592 and 666.
  • Embodiment 5 An engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
  • Embodiment 7 The engineered dsRNA ligase polypeptide of Embodiment 5 or 6, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, XI 14, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221,
  • Embodiment 8 The engineered dsRNA ligase polypeptide of Embodiment 7, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T;
  • Embodiment 9 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-8, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158,
  • Embodiment 10 The engineered dsRNA ligase polypeptide of Embodiment 9, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D
  • Embodiment 11 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-10, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
  • Embodiment 12 The engineered dsRNA ligase polypeptide of Embodiment 11, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • Embodiment 13 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-12, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
  • Embodiment 14 The engineered dsRNA ligase polypeptide of Embodiment 13, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
  • Embodiment 15 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-14, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X36, X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.
  • Embodiment 16 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-15, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; and X221 is l.
  • Embodiment 17 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-16, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X218, X221 and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.
  • Embodiment 18 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-17, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
  • Embodiment 19 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-18, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285
  • Embodiment 20 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-19, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X19, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X19 is D; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285
  • Embodiment 21 The engineered dsRNA ligase polypeptide of any one of Embodiments 5-20, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X 53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L;
  • Embodiment 22 The engineered dsRNA ligase polypeptide of any of Embodiments 1- 21, wherein the engineered dsRNA ligase polypeptide comprises a purification tag.
  • Embodiment 23 The engineered dsRNA ligase polypeptide of Embodiment 22, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
  • Embodiment 24 The engineered dsRNA ligase polypeptide of Embodiment 22, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
  • Embodiment 25 A polypeptide immobilized on a solid material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide according to any one of Embodiments 1-24.
  • Embodiment 26 A polynucleotide encoding the engineered dsRNA ligase polypeptide of any one of Embodiments 1-24.
  • Embodiment 27 The polynucleotide of Embodiment 26, wherein the polynucleotide sequence is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
  • Embodiment 28 The polynucleotide of Embodiment 26, wherein the polynucleotide sequence is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143
  • Embodiment 29 An expression vector comprising the polynucleotide according to any one of Embodiments 26-28.
  • Embodiment 30 The expression vector of Embodiment 29, which comprises a plasmid, a cosmid, a bacteriophage or a viral vector.
  • Embodiment 31 A host cell comprising the polynucleotide of any one of Embodiments 26-28 or the expression vector of Embodiment 29 or 30, optionally wherein the host cell is E. coli.
  • Embodiment 32 A method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing the host cell according to Embodiment 31 and obtaining an engineered dsRNA ligase polypeptide from the culture.
  • Embodiment 33 A method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing the host cell according to Embodiment 31 and obtaining an engineered dsRNA ligase polypeptide from the culture.
  • An engineered dsRNA ligase catalyst obtainable by culturing the host cells according to Embodiment 31, or according to the method of Embodiment 32, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cell, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cell, an extract thereof, or isolated product of the extract.
  • Embodiment 34 A method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting:
  • Embodiment 35 The method of Embodiment 34, wherein the source of ATP comprises ATP.
  • Embodiment 36 The method of Embodiment 34 or 35, wherein the source of ATP comprises:
  • Embodiment 37 The method of Embodiment 36, wherein the PPK is selected from PPK12 or ajPAP.
  • Embodiment 38 The method of any one of Embodiments 36 or 37, wherein the method is performed using a sub-stoichiometric concentration of AMP and/or ATP.
  • Embodiment 39 The method of any one of Embodiments 36-38, wherein the polyphosphate is a polyphosphate salt.
  • Embodiment 40 The method of Embodiment 39, wherein the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
  • the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
  • Embodiment 41 The method of any one of Embodiments 34-40, wherein the divalent cation cofactor is Mg 2+ or Mn 2+ .
  • Embodiment 42 The method of any one of Embodiments 34-41, wherein the method is performed with a divalent cation concentration of 5-100 mM, optionally 30-50 mM.
  • Embodiment 43 The method of any one of Embodiments 34-42, further comprising a step of purifying the oligonucleotide.
  • Embodiment 44 Use of the engineered dsRNA ligase polypeptide according to any one of Embodiments 1-24 in the production of an oligonucleotide from two or more oligonucleotide fragments.
  • Embodiment 45 The method of any one of Embodiments 34-43 or the use of
  • Embodiment 44 wherein the oligonucleotide is up to 60 nucleotides in length.
  • Embodiment 46 The method of any one of Embodiments 34-43 or 45 or the use of Embodiment 44 or 45, wherein each of the oligonucleotide fragments are 4-16 nucleotides in length, optionally 6-9 nucleotides in length.
  • Embodiment 47 The method of Embodiment 34-43, 45 or 46 or the use of any one of Embodiments 44-46, wherein one or more of the oligonucleotide fragment(s) comprises one or two overhangs.
  • Embodiment 48 The method of any one of Embodiments 34-43 or 45-47 or the use of any one of Embodiments 44-47, wherein one or more of the oligonucleotide fragments comprises a chemical modification.
  • Embodiment 49 The method or use of Embodiment 48, wherein the chemical modification is selected from:
  • a modified backbone optionally selected from a phosphorothioate (e.g. chiral phosphorothioate) or methylphosphonate intemucleotide linkage;
  • a phosphorothioate e.g. chiral phosphorothioate
  • methylphosphonate intemucleotide linkage optionally selected from a phosphorothioate (e.g. chiral phosphorothioate) or methylphosphonate intemucleotide linkage
  • a modified nucleotide optionally selected from 2'-O-methyl (2’-0Me), 2'-flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-0-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O- dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O- DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g.
  • GalNAc N- Acetylgalactosamine
  • Embodiment 50 A composition comprising: i. the engineered dsRNA ligase polypeptide according to any one of Embodiments 1- 24; ii. a source of ATP; and iii. a divalent cation.
  • Embodiment 51 The composition of Embodiment 50, further comprising two or more oligonucleotide fragments.
  • Embodiment 52 A kit comprising: i. the engineered dsRNA ligase polypeptide according to any one of Embodiments 1- 24; ii. a source of ATP; iii. a divalent cation; and iv. instructions for use in a method of producing an oligonucleotide from two or more oligonucleotide fragments.
  • Embodiment 53 The composition of Embodiment 50 or 51 or the kit of Embodiment 52, wherein the source of ATP comprises ATP.
  • Embodiment 54 The composition of any one of Embodiments 50, 51 or 53 or the kit of Embodiment 52 or 53, wherein the source of ATP comprises:
  • Embodiment 55 The composition or kit of Embodiment 54, wherein the PPK is selected from PPK 12 or ajPAP.
  • Embodiment 56 The composition of any one of Embodiments 50, 51 or 53-55 or the kit of any one of Embodiments 52-55, wherein the polyphosphate is a polyphosphate salt.
  • Embodiment 57 The composition or kit of Embodiment 56, wherein the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
  • Embodiment 58 The composition of any one of Embodiments 50, 51 or 53-57 or the kit of any one of Embodiments 52-57, wherein the divalent cation cofactor is Mg 2+ or Mn 2+ .
  • ppm parts per million
  • M molar
  • mM millimolar
  • uM and pM micromolar
  • nM nanomolar
  • mol molecular weight
  • gm and g gram
  • mg milligrams
  • ug and pg micrograms
  • L and 1 liter
  • ml and mb milliliter
  • cm centimeters
  • mm millimeters
  • um and pm micrometers
  • coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FIOP (fold improvements over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Coming (Coming, Inc., Palo Alto, CA); Dow Coming (Dow Coming, Corp., Midland, MI); and Gene Oracle (Gene Oracle, Inc., Mountain View, CA).
  • CGSC Coli Genetic Stock Center
  • HTP high throughput
  • HPLC high pressure liquid chromatography
  • FIOP fold improvements over positive control
  • Microfluidics Microfluidics (Microfluidics, Corp., Westwood, MA);
  • RNA (1) sequences of the oligonucleotides referred to in parentheses (e.g. “siRNA (1)” and “oligonucleotide (2)") throughout the Examples are provided in Table 1.
  • Polynucleotides encoding the polypeptides having ligase activity were cloned into the pCKl 10900 vector system (See e.g., US Pat. App. No. 2006/0195947A1 FIG. 3 which is hereby incorporated by reference in its entirety) and subsequently expressed in E. coli W311O 7n/A under the control of the lac promoter.
  • the expression vector also contained the Pl 5a origin of replication and the chloramphenicol (CAM) resistance gene.
  • E. coli W311O 7IMA cells were transformed with the pCKl 10900 plasmid containing the ligase-encoding genes.
  • Transformed cells were plated out on Lysogeny broth (LB) agar plates containing 1% glucose and 30 pg/mL CAM, and grown overnight at 37° C. Subsequently single colonies were inoculated into 25 mL of LB supplemented with 30 pg/mL CAM and 1% glucose in a 250 ml baffled shake flask. The culture was grown overnight (16-20 hours and optical density (ODeoo) >3.8) in an incubator at 37°C, with shaking at 250 rpm.
  • IPTG isopropyl-P-D-thiogalactoside
  • the cell pellet was resuspended in 30 mb of 50 mM Tris-buffer at pH 7.5 and lysed using a LM20 MICROFLUIDIZER® processor system (Microfluidics). Cell debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. Ligase enzymes were then isolated from the clarified lysate using standard techniques known in the art, including immobilized metal affinity chromatography.
  • siRNA (1) comprising of oligonucleotides (2) and (3)
  • siRNA (4) comprising of oligonucleotides (3) and (5).
  • Oligonucleotide (5) has the same sequence as oligonucleotide (2) but does not contain a 3’- GalNAc moiety.
  • siRNAs (1) and (4) and oligonucleotides (2), (3) and (5) are depicted in Figure 1; and the sequences of oligonucleotides (2), (3) and (5) are provided in Table 1.
  • SEQ ID NO: 2 for the production of siRNA (1) was subsequently confirmed using multiple enzyme preparations including isolated enzyme (example 1), clarified lysate (example 4) and shake flask powder (SFP; example 5).
  • isolated enzyme example 1
  • clarified lysate example 4
  • shake flask powder SFP; example 5
  • Single colonies were picked in a 96-well format and grown in 190 pL LB media containing 1% glucose and 30 pg/mL CAM, at 30°C, 200 rpm, and 85% humidity. Following overnight growth, 20 pL of the grown cultures were transferred into a deep well plate containing 380 pL of TB media with 30 pg/mL CAM. The cultures were grown at 30°C, 250 rpm, with 85% humidity for approximately 2.5 hours. When the ODeoo of the cultures reached 0.4-0.8, expression of the ligase gene was induced by the addition of IPTG to a final concentration of 1 mM. Following induction, growth continued for 18-20 hours at 30°C, 250 rpm with 85% humidity. Cells were harvested by centrifugation at 4,000 rpm and 4°C for 10 minutes; the supernatant was then discarded. The cell pellets were stored at -80°C until ready for use.
  • the cell pellets Prior to performing the assay, the cell pellets were thawed and resuspended in 300 pL of lysis buffer (containing 1 g/L lysozyme, 0.5 g/L PMBS and 0.1 pL/mL or 0.2U/ml of commercial DNAse (New England BioLabs, M0303L) in 50 mM Tris-buffer at pH 7.5.
  • the plates were agitated with medium-speed shaking for 2.5 hours on a microtiter plate shaker at room temperature. The plates were then centrifuged at 4,000 rpm for 10 minutes at 4°C, and the clarified supernatants were used in the HTP assay reaction for activity determination as described in the following examples.
  • Shake-flask procedures can be used to generate engineered dsRNA ligase polypeptide shake-flask powders (SFP), which are useful for secondary screening assays and/or use in the biocatalytic processes described herein.
  • Shake flask powder preparation of enzymes provides a more concentrated preparation of the engineered enzyme, as compared to the cell lysate used in HTP assays.
  • Clarified lysate produced according to example 1 was collected, frozen at -80°C, and then lyophilized, using standard methods known in the art. Lyophilization of frozen clarified lysate provides a dry SFP comprising crude wild-type or engineered dsRNA ligase polypeptide.
  • Table 6-1 HPUC method 1 used for activity determination.
  • HPLC method 2 (Table 6-2) was developed from HPLC method 1 (Table 6-1) to improve the separation between the product oligonucleotide (3) and the substrate oligonucleotide (12).
  • Table 6-2 HPLC method 1 used for activity determination.
  • RF-MS RapidFire® Mass Spectrometry
  • Table 6-3 The activity improvements of the engineered dsRNA ligases of Example 12 were also analyzed with RapidFire® Mass Spectrometry (RF-MS) using the method described in Table 6-3.
  • RF-MS aims to reduce the analytical time compared to HPLC analysis.
  • the selective detection of product oligonucleotides (2) and (3) is obtained by the specific masses of each product oligonucleotide analyzed under the multi-single ion monitoring (SIM) mode.
  • SIM multi-single ion monitoring
  • Relative dsRNA ligase activity is determined by comparing the sum of the MS signal of the five specific masses (given in Table 6-3) corresponding with each product oligonucleotide (2) and (3).
  • Table 6-3 RF-MS method used for activity determination.
  • the engineered polynucleotide (SEQ ID NO: 1) encoding the polypeptide with dsRNA ligase activity of SEQ ID NO: 2 was used to generate the engineered polypeptides of Table 7-
  • polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrate oligonucleotides (6-7, 9-12) as compared to the starting polypeptide.
  • Some polypeptides displayed improved product formation of either oligonucleotide product (2) or (3), or both oligonucleotide products (2) and (3) compared to the starting polypeptide as noted in Table 7-1.
  • the sequences of oligonucleotides (2), (3), (6), (7) and (9-12) are provided in Table 1.
  • the engineered polypeptides having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO:
  • the enzyme assays were carried out in 96-well PCR plates, in 50 pL total reaction volume per well.
  • the reactions contained 2.5 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 100 pM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.5, 1 mM ATP, 10 mM MgCh and 5 mM DTT.
  • the reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 2 h.
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 4 to 106 comprise an even numbered sequence identifier of SEQ ID NOs: 304 to 406, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 4 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 304.
  • the position of a given mutation is provided relative to SEQ ID NO: 2 which includes (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669 and (ii) the wild-type dsRNA ligase polypeptide of SEQ ID NO: 302.
  • the position of a given mutation relative to SEQ ID NO: 302 i.e. the wild-type dsRNA ligase polypeptide without the purification tag
  • position X251 of SEQ ID NO: 2 corresponds to position X237 of SEQ ID NO: 302.
  • polypeptide from example 7 SEQ ID NO: 69 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 70 was used to generate the engineered polypeptides of Table 8-1.
  • These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide.
  • polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 8-1.
  • the engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 70, as described below together with the analytical method described in Table 6-1.
  • Directed evolution began with the polynucleotide set forth in SEQ ID NO: 69.
  • Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
  • the enzyme assays were carried out in 96-well PCR plates, in 50 pL total reaction volume per well.
  • the reactions contained either 1.25 or 2.5 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 100 pM (each) substrate oligonucleotides (6-7, 9- 12), 50 mM Tris-buffer at pH 7.5, 1 mM ATP, 10 mM MgCh and 5 mM DTT.
  • the reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 2 h.
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 108 to 216 comprise an even numbered sequence identifier of SEQ ID NOs: 408 to 516, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 108 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 408.
  • polypeptide from example 8 SEQ ID NO: 187 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 188 was used to generate the engineered polypeptides of Table 9-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide.
  • polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 9- 1.
  • the engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 188, as described below together with the analytical method described in Table 6-2.
  • the enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well.
  • the reactions contained 20 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 1 mM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.0, 10 mM ATP, 20 mM MgCh, 5 mM DTT and 10 % (v/v) DMSO.
  • the reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 2 h.
  • the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate.
  • the plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 450 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were further diluted by transferring 50 pL of the diluted sample into a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0).
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 218 to 246 comprise an even numbered sequence identifier of SEQ ID NOs: 518 to 546, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 218 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 518.
  • polypeptide from example 9 SEQ ID NO: 225 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 226 was used to generate the engineered polypeptides of Table 10-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide.
  • polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 10-1.
  • the engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 226, as described below together with the analytical method described in Table 6-2.
  • Directed evolution began with the polynucleotide set forth in SEQ ID NO: 225.
  • Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
  • the enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well.
  • the reactions contained 2.5 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 1 mM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.0, 10 mM ATP, 20 mM MgCh, 5 mM DTT and 10 % (v/v) DMSO.
  • the reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
  • the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate.
  • the plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 450 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were further diluted by transferring 50 pL of the diluted sample into a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0).
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 248 to 282 comprise an even numbered sequence identifier of SEQ ID NOs: 548 to 582, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 248 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 548.
  • polypeptide from example 10 SEQ ID NO: 277 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 278 was used to generate the engineered polypeptides of Table 11-1.
  • These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide.
  • polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 11-1.
  • the engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 278, as described below together with the analytical method described in Table 6-2. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 277.
  • the enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well.
  • the reactions contained 10 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 5 mM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.0, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO.
  • the reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
  • the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate.
  • the plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were further diluted by transferring 50 pL of the diluted sample into a deep well 96-well plate containing 450 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were diluted a third time by transferring 160 pL of the diluted sample into a deep well 96-well plate containing 640 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were diluted a final time by transferring 75 pL of the diluted sample into a shallow well 96-well plate containing 75 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 278 are shown in Table 11-1.
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 284 to 300 comprise an even numbered sequence identifier of SEQ ID NOs: 584 to 600, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 284 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 584.
  • polypeptide from example 11 SEQ ID NO: 287 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 288 was used to generate the engineered polypeptides of Table 12-1.
  • These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrate oligonucleotides (6-7, 9-12) as compared to the starting polypeptide.
  • polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide, as noted in Table 12- 1. Furthermore, some polypeptides displayed improved thermal stability, resulting in higher residual activity following incubation of the dsRNA ligase solution at 30 °C for 1 h prior to setting up the reaction (Table 12-2).
  • the engineered polypeptides having the amino acid sequences of even- numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 288, as described below together with the analytical method described in Table 6-3. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 287.
  • the enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well.
  • the cells were lysed according to Example 4, however 100 mM MOPS-buffer at pH 7.2 was used in place of 50 mM Tris at pH 7.5.
  • the cell lysates were either undiluted or diluted 1 : 1 in 100 mM MOPS buffer, pH 7.2 and incubated at 30 °C and 4 °C respectively for 1 h.
  • the reactions contained a final dsRNA ligase concentration of 40 % (v/v) for lysate incubated at 30 °C and 20% (v/v) for lysate incubated at 4 °C.
  • the ligation reactions contained 3 mM (each) substrate oligonucleotides (6-7, 9-12), 100 mM MOPS-buffer at pH 7.2, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO.
  • the reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
  • the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate.
  • the plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were further diluted by transferring 20 pL of the diluted sample into a deep well 96-well plate containing 180 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were diluted a third time by transferring 30 pL of the diluted sample into a deep well 96-well plate containing 150 pL of 5 mM EDTA solution (pH 7.0).
  • the samples were analyzed via RF-MS to determine the activity of the enzyme variants using the analytical method described in Table 6-3.
  • Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 288 following pre-incubation at 4 °C are shown in Table 12-1.
  • Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 288 following pre-incubation at 30 °C are shown in Table 12-2.
  • the engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 602 to 634 comprise an even numbered sequence identifier of SEQ ID NOs: 636 to 668, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)).
  • SEQ ID NO: 602 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 636.
  • polynucleotides SEQ ID NO: 1 encoding for the wild-type dsRNA ligase from Bacteriophage RB69, Uniprot ID: Q7Y4V8 with the SEQ ID NO: 2 and the engineered polynucleotides SEQ ID NO: 287, SEQ ID NO: 289 and SEQ ID NO: 291 encoding for the most improved variants from example 11 with polypeptide sequences SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292, have been used for SFP production as described in example 5.
  • the catalytic activity to convert the substrates oligonucleotides (6-7, 9-12) to the desired siRNA product (1) was evaluated under two reaction conditions: Condition 1 (50 mM Tris-buffer at pH 7.5, 1 mM ATP, 5 mM MgCh and 5 mM DTT, containing either 0 g/L, 0.0020 g/L, 0.0039 g/L, 0.0078 g/L, 0.0156 g/L, 0.0313 g/L, 0.0625 g/L, 0.125 g/L, 0.25 g/L, 0.5 g/L, 1 g/L, or 2 g/L of SFP), and Condition 2 (50 mM Tris-buffer at pH 7.0, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO containing either 0 g/L, 0.0049 g/L, 0.0098 g/L, 0.0195 g/L, 0.0391
  • reaction plate 1 was incubated for 2 h and reaction plate 2 was incubated for 24 h.
  • reaction plate 1 was diluted 40 x and reaction plate 2 was diluted 2 400 x.
  • the samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2.
  • Comparative data in Figures 2A and 2B show the relative peak area % of siRNA (1) present in the reaction samples assayed under conditions 1 and 2 respectively. Under both conditions 1 and 2, the polypeptides SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292 exhibit improved dsRNA ligase activity over the wild-type polypeptide SEQ ID NO: 2.
  • polynucleotides SEQ ID NO: 1 encoding for the wild-type dsRNA ligase from Bacteriophage RB69, Uniprot ID: Q7Y4V8 with the SEQ ID NO: 2 and the engineered polynucleotide SEQ ID NO: 287 and SEQ ID NO: 631 encoding for the most improved variant from example 11 and example 12 with polypeptide sequences SEQ ID NO: 288 and SEQ ID NO: 632 respectively, have been used for SFP production as described in example 5.
  • the catalytic activity to convert the substrate oligonucleotides (6-7, 9-12) to the desired siRNA product (1) and the thermostability of the two enzymes was evaluated by incubating a stock solution of the SFP for 4 h at either 4 or 37 °C prior to setting up the following ligation reaction: 6 mM (each) substrate oligonucleotides (6-7, 9-12), 100 mM MOPS-buffer at pH 7.2, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO.
  • the ligation reactions contained, either 0 g/L, 0.156 g/L, 0.313 g/L, 0.625 g/L, 1.25 g/L, 2.5 g/L, 5 g/L, or 10 g/L of SFP.
  • the enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well. The reaction plate was heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
  • the plate was subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added SFP.
  • the plate was then centrifuged at 4,000 rpm for 5 min.
  • a 50 pL aliquot of the supernatant from each well of each plate was removed and subsequently diluted 400 x in 50 mM EDTA solution (pH 7.0).
  • the samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2.
  • Comparative data in Figure 3A shows the relative peak area % of siRNA (1) present in the reaction samples.
  • Figure 3B shows the residual enzyme activity following pre -incubation of the SFP at 37 °C for 4 h, expressed relative to the ligation activity of the SFP pre-incubated at 4 °C for 4 h.
  • the polypeptides SEQ ID NO: 632 exhibits improved dsRNA ligase activity and thermostability over the wild-type polypeptide SEQ ID NO: 2 and engineered polypeptide SEQ ID NO: 288.
  • Table 13 provides a summary of the nucleic acid and amino acid sequences of the wild-type and engineered dsRNA ligase sequences described herein.
  • the purification tag used in the Examples and reference in table 13 is the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669).

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Abstract

The present disclosure relates to the field of biotechnology, in particular to engineered double-stranded RNA (dsRNA) ligases and their application in industrial biocatalysis. The present disclosure also relates to a process of producing an engineered dsRNA ligase, and to a method for producing an oligonucleotide by contacting oligonucleotide fragments with an engineered dsRNA ligase.

Description

ENGINEERED DOUBLE-STRAND RNA LIGASES AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to, and the benefit of, EP Application No. 22215201.9, filed on December 20, 2022, the content of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on 2 December 2023, is named PAT059445-WO-PCT_SL.xml and is 1.24MB in size.
Technical field
The present disclosure relates to the field of biotechnology, in particular to engineered double-stranded RNA (dsRNA) ligases and their application in industrial biocatalysis. The present disclosure also relates to a process of producing an engineered dsRNA ligase, and to a method for producing an oligonucleotide by contacting oligonucleotide fragments with an engineered dsRNA ligase.
Background art
Therapeutic oligonucleotides, including small interfering RNA (siRNA) and inhibitory antisense oligonucleotides (ASOs) have the potential to treat a diverse range of life-threatening diseases. In recent years there has been a significant increase in the number of approved oligonucleotide-based drugs, and a large rise in the number of therapeutic oligonucleotides under clinical investigation (Roberts, T. C., Langer, R. & Wood, M. J. A. Nature Reviews Drug Discovery 2020 19:10 19, 673-694 (2020)).
In support of green synthesis initiatives throughout the pharmaceutical industry, there is a significant need for next-generation oligonucleotide synthesis methods that are both sustainable and economical at the scale required to reach wider patient populations (Mishra, M. et al. Current Research in Green and Sustainable Chemistry 4, (2021)).
To this end, biocatalysis is being more frequently applied in the manufacture of active pharmaceutical ingredients (APIs) since enzymes are capable of highly selective transformation under mild reaction conditions and in aqueous media (Mann, G. & Stanger, F. V. Chimia (Aarau) 74, 407-417 (2020)). The biocatalysis of short oligonucleotide fragments offers a sustainable and economical alternative to the solid phase chemical synthesis of full- length therapeutic oligonucleotides currently used.
Shorter oligonucleotides can be synthesized more easily and with higher purities than longer oligonucleotides, simplifying downstream processing and reducing solvent waste. These short oligonucleotide fragments can then be combined using nucleic acid ligases to produce oligonucleotide products. Nucleic acid ligases have shown remarkable tolerance towards unnatural DNA/RNA containing pharmaceutically relevant chemical modifications (Kestemont, D., Herdewijn, P. & Renders, M. Curr Protoc Chem Biol 11, e62 (2019); Kestemont, D. et al. Chemical Communications 54, 6408-6411 (2018); and Nandakumar, J. & Shuman, S. Molecular Cell 16, 211-221 (2004)), and the use of a dsRNA ligase to synthesize an siRNA product, starting from short fragments (< 9 nts), containing extensive chemical modification, including 2’-OMe, 2’-F modified nucleotides, phosphorothioate backbone modified nucleotides and a terminal fragment that is functionalized with a bulky N- acetyl galactosamine (GalNAc) moiety has previously been described (Mann, G. et al. Tetrahedron Letters 93, 153696 (2022)).
To achieve cost-effective and sustainable industrial scale biocatalysis of oligonucleotides, enzymes exhibiting high ligase activity are required. There exists an urgent and unmet need for engineered ligase enzymes, which exhibit improved ligase activity relative to wild-type enzymes. There is also an unmet need for biocatalytic methods of producing oligonucleotides from oligonucleotide fragments.
Brief description of the drawings
Figure 1. dsRNA ligase catalyzed ligation of: (A) oligonucleotide fragments 6:9; 7: 10 and 11: 12 to generate oligonucleotide 2:3 (= siRNA 1); and (B) oligonucleotide fragments 6:9, 7: 10 and 8: 11 to generate oligonucleotide 5:3 (= siRNA 4). The sequences of oligonucleotides 2, 3 and 5-12 are provided in Table 1.
Figure 2. Comparative data showing the relative peak area % of siRNA (1) present in the reaction samples comprising different concentrations of the wild-type enzyme (SEQ ID NO: 2) and engineered enzymes (SEQ ID NOs: 288, 290 and 292) assayed under: (A) condition 1; and (B) condition 2, as described in Example 13. Enzyme concentration is provided as g/L of shake-flask powder (SFP) produced by lyophilization of frozen clarified lysate as described in the Examples.
Figure 3. (A) Comparative data showing the relative peak area % of siRNA (1) present in the reaction samples comprising different concentrations of wild-type enzyme (SEQ ID NO: 2) and engineered enzymes (SEQ ID NOs: 288 and 632) following pre-incubation of the enzyme at 4 °C or 37 °C for 4 h. (B) Comparative data showing residual enzyme activity following pre-incubation of the SFP at 37 °C for 4 h, expressed relative to the ligation activity of the SFP pre-incubated at 4 °C for 4 h. Enzyme concentration is provided as g/L of shake-flask powder (SFP) produced by lyophilization of frozen clarified lysate as described in the Examples.
Summary of the disclosure
The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides. The present disclosure also provides gene sequences of engineered polypeptides, recombinant expression vectors comprising the genes, engineered host strains and efficient methods for the production thereof, as well as reaction processes for the biocatalysis of oligonucleotides using engineered polypeptides.
The engineered double -stranded RNA (dsRNA) ligase polypeptides described herein have improved catalytic activity as compared to the wild-type dsRNA ligase from which they are derived. Through substitutions and/or deletions of amino acid residues in directed evolution processes, the engineered polypeptides provided herein were derived from a wildtype dsRNA ligase from Bacteriophage RB69. The wild-type dsRNA ligase consists of 332 amino acids and has the amino acid sequence shown in SEQ ID NO: 302 (also accessible under accession number Q7Y4V8 in UniProt).
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,
528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,
564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and
600; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302. The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,
528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,
564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,
600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,
528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,
564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,
600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not the comprise the amino acid sequence of SEQ ID NO: 302. The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,
464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,
500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,
536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570,
572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,
464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,
500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570,
572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640,
642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having
(i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,
528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,
564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,
600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E ; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 666. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 666; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10) of the following amino acid residues: X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 370; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 488; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, or all 3) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 526; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 578; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) of the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 588 or 590; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) of the following amino acid residues: X15 is D or E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 592; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) of the following amino acid residues: X19 is D; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666 and 668. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 642, 646, 664, and 666.
The disclosure also provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
In some embodiments, the ligation reaction conditions include about 1 pM to about 10 mM oligonucleotide fragment, a source of ATP, about 5 mM to about 100 mM divalent cation, and about 0.5 g/L to about 10 g/L engineered dsRNA ligase polypeptide, pH of about 4.0 to about 8.0, and temperature of about 10 °C to about 50°C. In some embodiments, the source of ATP comprises ATP, optionally a stoichiometric excess of ATP. In some embodiments, the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, or R; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity. In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, or all 4) amino acid residues selected from: X36, X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, or all 3) amino acid residues selected from: X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, or all 3) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, or all 4) amino acid residues selected from: X39, X218, X221 and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, or all 4) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) of the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) of the following amino acid residues: X15 is E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) amino acid residues selected from: X19, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all 9) of the following amino acid residues: X19 is D; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10) amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10) of the following amino acid residues: X15 is D; X39 is A; X 53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
In some embodiments, the engineered dsRNA ligase polypeptide comprises a purification tag. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,
146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,
182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,
218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,
254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,
290, 292, 294, 296, 298 and 300. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,
186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,
222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,
258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628,
630, 632, and 634.
The disclosure also provides a polypeptide immobilized on a solid material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide described herein. The disclosure also provides a polynucleotide encoding the engineered dsRNA ligase polypeptide described herein.
In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,
205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,
241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,
277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 303, 305, 307, 309, 311, 313,
315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,
351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,
387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421,
423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457,
459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493,
495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529,
531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565,
567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, and 599.
In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667.
In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from: (a) SEQ ID NOs: 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365,
367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401,
403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,
439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473,
475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509,
511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545,
547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581,
583, 585, 587, 589, 591, 593, 595, 597, 599, 635, 637, 639, 641, 643, 645, 647, 649, 651,
653, 655, 657, 659, 661, 663, 665, and 667; and/or (b) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,
149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,
185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,
221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,
257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291,
293, 295, 297, 299, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627,
629, 631, and 633.
The disclosure also provides an expression vector comprising the polynucleotide described herein. In some embodiments, the vector comprises a plasmid, a cosmid, a bacteriophage or a viral vector.
The disclosure also provides a host cell comprising the polynucleotide described herein or the expression vector described herein. In some embodiments, the host cell is E. coli.
The disclosure also provides a method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing the host cell described herein and obtaining an engineered dsRNA ligase polypeptide from the culture.
The disclosure also provides an engineered dsRNA ligase catalyst obtainable by culturing the host cells described herein, or according to the method described herein, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cell, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cell, an extract thereof, or isolated product of the extract.
The disclosure further provides a method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting: (i) two or more oligonucleotide fragments; (ii) an engineered dsRNA ligase polypeptide described herein;
(iii) a source of ATP; and (iv) a divalent cation; to obtain an oligonucleotide.
In some embodiments, the source of ATP comprises ATP.
In some embodiments, the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP. In some embodiments, the PPK is selected from PPK 12 or ajPAP. In some embodiments, the method is performed using a sub-stoichiometric concentration of AMP and/or ATP.
In some embodiments, the polyphosphate is a polyphosphate salt. In some embodiments, the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
In some embodiments, the divalent cation cofactor is Mg2+ or Mn2+.
In some embodiments, the method is performed with a divalent cation concentration of 5-100 mM, optionally 30-50 mM.
In some embodiments, the method further comprises a step of purifying the oligonucleotide.
The disclosure also provides use of the engineered dsRNA ligase polypeptide described herein in the production of an oligonucleotide from two or more oligonucleotide fragments.
In some embodiments, the oligonucleotide is up to 60 nucleotides in length.
In some embodiments, each of the oligonucleotide fragments are 4-16 nucleotides in length, optionally 6-9 nucleotides in length.
In some embodiments, one or more of the oligonucleotide fragment(s) comprises one or two overhangs.
In some embodiments, one or more of the oligonucleotide fragments comprises a chemical modification. In some embodiments, the chemical modification is selected from: (a) a modified backbone, optionally selected from a phosphorothioate (e.g. chiral phosphorothioate) or methylphosphonate intemucleotide linkage; (b) a modified nucleotide, optionally selected from 2'-O-methyl (2’-OMe), 2'-flouro (2’-F), 2'-deoxy, 2'-deoxy-2’- fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O- dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g. mesyl phosphoramidate), 2',3'-seco nucleotide mimic, 2'-F-arabino nucleotide, abasic nucleotide, 2'- amino modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, vinylphosphonate (e.g. 5’ vinylphosphonate), and cyclopropyl phosphonate deoxyribonucleotide; and/or (c) conjugation to a ligand, optionally wherein the ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives.
The disclosure also provides a composition comprising: i. the engineered dsRNA ligase polypeptide described herein; ii. a source of ATP; and iii. a divalent cation. In some embodiments, the composition further comprises two or more oligonucleotide fragments.
The disclosure also provides a kit comprising: i. the engineered dsRNA ligase polypeptide described herein; ii. a source of ATP; iii. a divalent cation; and iv. instructions for use in a method of producing an oligonucleotide from two or more oligonucleotide fragments.
In some embodiments, the source of ATP comprises ATP.
In some embodiments, the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
In some embodiments, the PPK is selected from PPK 12 or ajPAP.
In some embodiments, the polyphosphate is a polyphosphate salt.
In some embodiments, the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
In some embodiments, the divalent cation cofactor is Mg2+ or Mn2+.
Definitions
Unless expressly defined otherwise, technical and scientific terms used in this disclosure have the meanings that are commonly understood by people skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
As used throughout this disclosure, articles such as “a” and “an” refer to one or more than one (at least one) of the grammatical object of the article.
The term “and/or” means either “and” or “or” unless indicated otherwise.
As used herein, the term “about” typically refers to the value which immediately follows the term ‘about’. For example, “about 15 or more nucleotides” typically refers to 15 or more nucleotides. In some embodiments, the term “about” embraces values which are +/- 1, 2 or 3 of the stated value. For example, “about 15 or more nucleotides” may refer to 15+/-3 nucleotides, e.g. 12, 13, 14, 15, 16, 17 or 18 nucleotides. The terms “double-stranded RNA ligase” and “dsRNA ligase” are used interchangeably herein to refer to an enzyme having dsRNA ligase activity. A dsRNA ligase polypeptide may also be referred to herein as a “dsRNA ligase catalyst”.
A dsRNA ligase of the invention is an ATP-dependent nucleic acid ligase. dsRNA ligase activity as used herein typically involves the ATP-dependent formation of a covalent bond between the 3 ’-OH of a ribonucleotide and the 5’-PO4 of a ribonucleotide or deoxyribonucleotide via the following steps: (1) dsRNA ligase reacts with ATP to form a covalent dsRNA ligase-AMP intermediate and release pyrophosphate; (2) AMP is transferred from the dsRNA ligase-AMP intermediate to the 5 ’-phosphate of a 3’ oligonucleotide fragment forming an adenylated oligonucleotide intermediate; and (3) the 3 ’-OH of a 5’ oligonucleotide fragment attacks the 5 ’ phosphate of the adenylated intermediate resulting in the formation of a phosphodiester bond and the release of AMP.
The stoichiometric concentration of cofactor is the theoretical concentration required to achieve complete ligation in a given ligation reaction. The skilled person can readily derive the stoichiometric concentration of ATP required to achieve complete ligation based on the concentration of oligonucleotide fragments and the number of ligation reactions required to produce the oligonucleotide product. For example, a ligation reaction using 1 mM substrate which requires four ligation reactions has a stoichiometric ATP concentration of 4 mM. A stoichiometric excess of ATP can help ensure that complete ligation is achieved. In some embodiments, the stoichiometric excess is at least 105% of the theoretical stoichiometric concentration of ATP required to achieve complete ligation, e.g. at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.
The terms "engineered dsRNA ligase", "engineered dsRNA ligase polypeptide", "improved dsRNA ligase polypeptide", and "engineered polypeptide" are used interchangeably herein.
As used herein, the term “oligonucleotide” refers to a nucleic acid, typically comprising up to 100 nucleotides. As used herein, the term “oligonucleotide product” refers to an oligonucleotide formed by the ligation of two or more oligonucleotide fragments by a dsDNA ligase described herein. Oligonucleotide products are also referred to herein simply as oligonucleotides. It will be understood that oligonucleotide products described herein comprise RNA. It will also be understood that oligonucleotide products described herein comprise a double-stranded region. In some embodiments, oligonucleotide products described herein comprise RNA and DNA. For example, a portion of the oligonucleotide product may be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.
The term “therapeutic oligonucleotide” refers to an oligonucleotide that can provide a therapeutic effect, e.g. by interacting with a biomolecule and/or by regulating gene expression. Therapeutic oligonucleotides include, but are not limited to, RNA interference (RNAi) agents and antisense oligonucleotides (ASO). RNAi is a post-transcriptional, targeted gene-silencing technique that uses RNAi agents to degrade messenger RNA (mRNA) containing the same sequence as the RNAi agent. ASOs are single-stranded nucleic acids that can be used to target mRNA derived from a gene of interest. ASOs can alter gene expression via a number of mechanisms including direct steric blockage of mRNA and ribonuclease H (RNase H) mediated degradation of mRNA.
RNAi agents include, as non-limiting examples, siRNAs (small interfering RNAs), dsRNAs (double-stranded RNAs), shRNAs (short hairpin RNAs) and miRNAs (micro RNAs). RNAi agents also include, as additional non-limiting examples, locked nucleic acid (LNA), Morpholino, UNA, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA) and fluoro-arabinonucleic acid (FANA). RNAi agents also include molecules in which one or more strands are a mixture of RNA, DNA, LNA, Morpholino, UNA (unlocked nucleic acid), TNA, GNA, and/or FANA. As a non-limiting example, one or both strands of an RNAi agent could be, for example, RNA, except that one or more RNA nucleotides is replaced by DNA, LNA, Morpholino, UNA, TNA, GNA, and/or FANA, etc. In some embodiments, one or both strands of the RNAi agent can be nicked, and both strands can be the same length, or one strand can be shorter than the other. The oligonucleotide of the invention may be any of the RNAi agents described herein.
The term “oligonucleotide fragment” herein refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide (or oligonucleotide product). Each oligonucleotide fragment corresponds to a portion of the oligonucleotide product. Oligonucleotide fragments may be referred to herein as “substrates” of the ligation reaction.
As described above, dsRNA ligase activity involves the ligation of a 5’ oligonucleotide fragment to a 3 ’ oligonucleotide fragment. In the context of oligonucleotide fragments, the prefixes 5’ and 3’ refer to the relative position of each oligonucleotide fragment in the oligonucleotide product after ligation, wherein the 5’ oligonucleotide fragment is located upstream of the 3 ’ oligonucleotide fragment (when the oligonucleotide product is presented in the 5’ to 3’ direction). As used herein, a “5’ oligonucleotide fragment” typically comprises a 3’ terminal ribonucleotide having a 3 ’-hydroxyl group. As used herein, a “3’ oligonucleotide fragment” comprises a 5’-phosphate, wherein the 5’ terminal nucleotide is a deoxyribonucleotide or a ribonucleotide.
It will be understood that, in some embodiments, an oligonucleotide fragment may be a 3’ oligonucleotide fragment and a 5’ oligonucleotide fragment (e.g. wherein ligation reactions occur at the 5’ and 3’ ends of the oligonucleotide fragment). For example, said oligonucleotide fragment may provide: (i) the 3’ oligonucleotide fragment in a ligation reaction with a 5’ oligonucleotide fragment; and (ii) the 5 ’ oligonucleotide fragment in a ligation reaction with a 3’ oligonucleotide fragment. For example, oligonucleotide fragment 7 in Figure 1A provides: (i) the 3’ oligonucleotide fragment in a ligation reaction with 5’ oligonucleotide fragment 6 and; (ii) the 5 ’ oligonucleotide fragment in a ligation reaction with 3 ’ oligonucleotide fragment 12 to provide oligonucleotide product 2.
A “terminal oligonucleotide fragment” herein refers to a nucleic acid that corresponds to an end (e.g. 5’ or 3’ end) portion of the oligonucleotide product. The 5’ terminal oligonucleotide fragment typically provides a 5’ oligonucleotide fragment for ligation to a 3’ oligonucleotide fragment. The 3’ terminal oligonucleotide fragment typically provides a 3’ oligonucleotide fragment for ligation to a 5 ’ oligonucleotide fragment. In some embodiments, the 5’ terminal oligonucleotide is ligated directly to the 3’ terminal oligonucleotide. In some embodiments, the 5 ’ terminal oligonucleotide and the 3 ’ terminal oligonucleotide are separated by one or more oligonucleotide fragments.
In some embodiments, oligonucleotide fragments described herein comprise RNA and DNA. For example, a portion of an oligonucleotide fragment may be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.
The term “overhang” or “nucleotide overhang” herein refers to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of a double -stranded oligonucleotide. In some embodiments, when a 3 '-end of one strand extends beyond the 5 '-end of the other strand, or vice versa, this forms a nucleotide overhang, e.g., the unpaired nucleotide(s) form the overhang. An overhang that is complementary to the overhang of a second oligonucleotide fragment may be referred to as a “sticky end". The oligonucleotide fragments described herein may have one or two sticky ends.
“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of a double-stranded oligonucleotide, i.e. , no nucleotide overhang. A “blunt ended” oligonucleotide or oligonucleotide fragment is an oligonucleotide that is double-stranded over its entire length, i. e. , no nucleotide overhang at either end of the molecule. Double -stranded nucleic acids comprise two anti-parallel and substantially complementary nucleic acid strands which are referred to as “sense” and “antisense” strands. In the context of double-stranded RNAi agents, the “antisense strand” refers to the strand of an RNAi which includes a region that is substantially complementary to a target sequence, e.g. an mRNA sequence. The “sense strand” refers to the strand of an RNAi that includes a region that is substantially complementary to a region of the antisense strand. The sense and antisense strands of an RNAi agent may be referred to as the passenger and guide strands, respectively.
Sequences that are “substantially complementary” may be fully complementary or may contain one or more mismatches upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application.
"Conversion" refers to the enzymatic transformation of a substrate to the corresponding product. "Percent conversion" or "conversion" refers to the percentage of oligonucleotide fragments that is converted to oligonucleotide product within a defined period of time under specified conditions. Thus, "enzymatic activity" or "activity" of a ligase can be expressed as the "percent conversion" of oligonucleotide fragments to oligonucleotide product.
Ideally to compare the activity between ligation reactions and account for natural variation in peak intensity between injections, the % conversion to product would be calculated for each sample analyzed using the following equation: 100
Figure imgf000022_0001
Whereby £p, £s and £i = the extinction coefficient of the product, substrate, and intermediate oligonucleotides respectively. In some instances, such as using the analytical method described herein, it is not possible to resolve all substrates, reaction intermediates and products. Therefore, the % conversion according to the above equation cannot be determined. However, in some instances, such as using the analytical method described herein, it is possible to resolve at least one substrate, reaction intermediate and product, such as well- defined GalNAc-containing oligonucleotides, including GalNAc containing substrate fragments (e.g. oligonucleotide (12) as used in the examples described herein), reaction intermediates (e.g. oligonucleotide (14) as demonstrated herein) and product strands (e.g. product oligonucleotide (2) as demonstrated herein). Therefore, a pseudo-% conversion can be calculated, denoted with arbitrary units (AU), which considers only these well resolved species according to the following equation:
Figure imgf000023_0001
whereby 8(2), 8(12) and 8<i4) are the extinction coefficient of oligonucleotides (2), (12) and (14) respectively. Using such a calculation an AU = 1.0 would imply that no more GalNAc- containing substrate or intermediate oligonucleotides are present in the reaction and that they have all be converted to GalNAc -containing product (2). In reality, for samples where AU = 1.0 the only other peak present in the chromatogram corresponds with the product oligonucleotide (2), and no other intermediates or starting materials can be identified. Furthermore, the ratio of the product oligonucleotides (2) and (3) are consistent with that of the authentic standard of siRNA product (1). Taken together, it can be concluded that AU = 1.0 is an approximation that is essentially equivalent to 100 % conversion.
"Improved enzyme properties" refers to an enzyme property that is better or more desirable for a specific purpose as compared to a reference dsRNA ligase such as a wild-type dsRNA ligase or another engineered dsRNA ligase under the same reaction conditions. Improved enzyme properties are exhibited by engineered dsRNA ligase polypeptides in this disclosure. The engineered dsRNA ligase polypeptides described herein exhibit increased enzyme activity (which can be expressed as a percentage of substrate conversion). Additional enzyme properties that may be improved include, but are not limited to, thermal stability, pH activity characteristics, cofactor requirements, and tolerance to inhibitors (e.g., reaction component, substrate or product inhibition).
An "isolated polypeptide" refers to a polypeptide that is substantially separated from other substances with which it is naturally associated, such as proteins, lipids, and polynucleotides. The term comprises polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., in host cells or in vitro synthesis). Engineered dsRNA ligase polypeptides may be present in the cell, in the cell culture medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the engineered dsRNA ligase polypeptide may be an isolated polypeptide.
"Wild-type" refers to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism that can be isolated from sources in nature, and which has not been intentionally modified by manual procedures. The polypeptide sequence of the wild-type dsRNA ligase described herein is provided by SEQ ID NO: 302. As used herein, the wild-type sequence may also comprise a purification tag and may be provided by SEQ ID NO: 2.
The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.
The terms "protein", "polypeptide" and "peptide" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.).
"Recombinant" or "engineered" when used with reference to, for example, a cell, nucleic acid or polypeptide, refers to a material or material corresponding to the native or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but produced or derived from synthetic material and/or by manipulation using recombinant techniques.
The abbreviations used for the genetically encoded amino acids are conventional and are as follows:
Figure imgf000024_0001
When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a-carbon, “D-Ala” and “L- Ala” designate D-alanine and L-alanine, respectively.
When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower-case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D-alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.
The abbreviations used for the genetically encoding nucleotides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleotides may be either ribonucleotides or 2 ’-deoxyribonucleotides. The nucleotides may be specified as being either ribonucleotides or 2 ’-deoxyribonucleotides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5’ to 3’ direction in accordance with common convention, and the phosphodiester bonds are not indicated.
The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of oligonucleotides featured in the present disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form Wobble base pairing with the target mRNA.
"Amino acid difference" or "residue difference" refers to the difference in amino acid residues at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in the reference sequence. The positions of amino acid differences are generally referred to herein as "Xn", where n refers to the corresponding position in the reference sequence on which the residue differences are based. For example, "a residue difference at position X6 as compared to SEQ ID NO: 302" refers to a difference in amino acid residue at the polypeptide position corresponding to position 6 of SEQ ID NO: 302. Thus, if the reference polypeptide of SEQ ID NO: 302 has a serine at position 6, then "a residue difference at position X2 as compared to SEQ ID NO: 302" refers to an amino acid substitution to any residue other than serine at the position of the polypeptide corresponding to position 6 of SEQ ID NO: 302.
The specific amino acid residue difference at the position may be indicated as "XnY" or “Xn is Y”, wherein "Xn" specifies the corresponding position in the reference sequence as described above, and "Y" is the single letter identifier of the residue present at that position in the engineered polypeptide. Specific amino acid differences may also be denoted by the conventional notation "AnY", where A is a single letter identifier of the residue in the reference sequence, "n" is the number of residue position in the reference sequence, and “Y” is the single letter identifier of the residue present at that position in the engineered polypeptide.
In some examples, an engineered polypeptide of this disclosure may comprise one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of specific positions at which residue differences are present relative to a reference sequence. In some embodiments, more than one amino acid residue can be used in a specific residue position of an engineered polypeptide, the various amino acid residues can be listed as alternatives, e.g. “X19 is Q or D”.
Deletion of an amino acid may be represented by e.g. “an amino acid sequence comprising Xn-” indicates that the amino acid sequence contains a deletion at the position corresponding to “Xn” in the reference sequence. "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids of the enzyme, or up to 20% of the total number of amino acids making up the reference enzyme while retaining the enzymatic activity of the engineered dsRNA ligase and/or retaining the improved properties of the engineered dsRNA ligase. Deletion may involve the internal portion and/or the terminal portion of the polypeptide. In various embodiments, deletions may include a contiguous segment or may be discontinuous.
In the context of the numbering for a given amino acid or polynucleotide sequence, "corresponding to," "reference to" or "relative to" refers to the numbering of the residues of a specified reference when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given sequence is designated with respect to the reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence such as an engineered dsRNA ligase can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although there are gaps, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.
"Reference sequence" refers to a defined sequence that is used as a basis for sequence comparison. The reference sequence may be a subset of a larger sequence, for example, a full-length gene or a fragment of a polypeptide sequence. In some embodiments, a "reference sequence" is a wild-type sequence. In some embodiments, a "reference sequence" is an engineered or altered sequences.
Methods of determining percentage sequence identity are known in the art. By way of example, when assessing sequence identity, a sequence having a defined number of contiguous nucleotides or amino acids may be aligned with a nucleic acid or peptide sequence (having the same number of contiguous nucleotides or amino acids) from the corresponding portion of a nucleic acid or peptide sequence disclosed herein. The percentage sequence identity can be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences, or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the sequence and multiplying the result by 100 to yield the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available to align two sequences. The optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, by the Homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Package) or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al. eds., Current Protocols, a Joint Venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining the percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information website. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold scores T when aligned with a word of the same length in the database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., Supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative scores are calculated using the parameters M (reward score for matched pair of residues; always> 0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quality X from its maximum achieved value; the cumulative score goes 0 or below, due to the accumulation of one or more negative -scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, the expected value (E) of 10, M = 5, N = -4, and a comparison of both strands as a default value. For amino acid sequences, the BLASTP program uses as defaults the word length (W) of 3, the expected value (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). Exemplary determination of sequence alignments and %sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using the default parameters provided.
It will be appreciated that, regardless of the percent sequence identity to a reference sequence, an engineered dsRNA ligase possesses dsRNA ligase activity.
"Suitable reaction conditions" refer to those conditions (e.g., enzyme loading, substrate loading, temperature, pH, etc.) in the reaction system, under which the substrate is converted to the desired product. Suitable reaction conditions can be readily identified by the person skilled in the art. Exemplary "suitable reaction conditions" are provided in the present disclosure and illustrated by examples. Engineered dsRNA ligase polypeptides
The disclosure provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,
528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,
564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and
600.
The disclosure provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668.
The disclosure provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,
492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,
528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562,
564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598,
600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668.
The disclosure also provides an engineered dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,
346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,
382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,
418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,
454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,
526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560,
562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596,
598, and 600, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure also provides an engineered dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure also provides an engineered dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,
346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380,
382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,
418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,
454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488,
490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524,
526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560,
562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596,
598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and
668, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,
464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,
500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
The disclosure provides an engineered double-stranded RNA (dsRNA) ligase polypeptide, which is a polypeptide of: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354,
356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,
464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,
500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,
536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570,
572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640,
642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, which comprises an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) a substitution, deletion, addition or insertion of one or more amino acid residues relative to said one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600, optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 636-668, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 636-668.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600 or 636-668, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to an even numbered sequence identifier of SEQ ID NOs: 304-600 or 636-668.
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 exhibit higher activity than that of SEQ ID NO: 302, as shown in the Examples. The dsRNA ligase polypeptides used in the Examples (represented by even numbered sequence identifiers of SEQ ID NOs: 4 to 300 and 602 to 634, respectively) comprise an even numbered sequence identifier of SEQ ID NOs: 304 to 600 and 636 to 668 and an N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 4 comprises: (i) the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 304. dsRNA ligase polypeptides represented by even numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 do not comprise the N-terminal purification tag represented by SEQ ID NO: 669.
The wild-type dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 302 (also accessible under UniProt accession number Q7Y4V8). SEQ ID NO: 2 comprises: (i) the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 302. It will be readily understood that SEQ ID NOs: 2 and 302 both comprise the wild-type dsRNA ligase polypeptide sequence and so both sequences may be referred to herein as the wild-type sequence.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590 and 592. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592 and 666. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 188, 226, 278, 288, 290 and 292. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 188, 226, 278, 288, 290, 292 and 632. SEQ ID NOs: 70, 188, 226, 278, 288, 290, 292 and 632 comprise: (i) an N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) an amino acid sequence provided by SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592 and 666, respectively.
In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 588. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666.
The disclosure also provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide produces at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions. In some embodiments, the ligation reaction conditions are as described herein.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 302, optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 302.
In some embodiments, the ligation reaction conditions include about 1 pM to about 10 mM oligonucleotide fragment, a source of ATP, about 5 mM to about 100 mM divalent cation, and about 0.5 g/L to about 10 g/L engineered dsRNA ligase polypeptide, pH of about 4.0 to about 8.0, and temperature of about 10 °C to about 50 °C. In some embodiments, the source of ATP is a stoichiometric concentration of ATP or a stoichiometric excess of ATP. In some embodiments, the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, XI 14, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the following amino acid residues: X6 is G ; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, or R; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11) amino acid residues selected from: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11) of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12) amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, or 12) of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more or three or more) amino acid residues selected from: X36, X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more or three or more) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X36, X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more) amino acid residues selected from: X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more) of the following amino acid residues: X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X39, X218 and X221; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more or three or more) amino acid residues selected from: X39, X218, X221, and X255; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more orthree or more) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X39, X218, X221, and X255; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, or seven or more) amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more, three or more, four or more, five or more, six or more, or seven or more) of the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more) amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g. two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more) of the following amino acid residues: X15 is D or E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at amino acid residues: X15, X39, X53, X218, X221, X237, X251, X255 and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X15 is D; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X15 is E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X 53 is Y; XI 85 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
In some embodiments, the engineered dsRNA ligase polypeptide comprises a purification tag. Purification tags are typically appended to polypeptides so that they can be purified from their crude biological source using an affinity technique. In some embodiments, the purification tag comprises a poly-histidine tag. Poly-histidine tags bind to matrices bearing immobilized metal ions and can be used to purify polypeptides by affinity chromatography. In some embodiments, the purification tag further comprises a protease recognition site for removal of the purification tag. In some embodiments, the protease recognition site comprises a Tobacco Etch Virus (TEV) protease recognition sequence. In some embodiments, the purification tag comprises the amino acid sequence MHHHHHHENLYFQS (SEQ ID NO: 669).
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,
222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,
258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298 and 300.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.
In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,
186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,
222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,
258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628,
630, 632, and 634.
Immobilization
The disclosure also provides a polypeptide immobilized on a solid support material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide disclosed herein.
Immobilization of a polypeptide by physical absorption typically involves the polypeptide being physically adsorbed or attached onto a solid support material. Adsorption can occur through weak non-specific forces such as van der Waals, hydrophobic interactions and hydrogen bonds. Physical adsorption may be achieved by soaking the support material in a solution of the polypeptide and incubating to allow time for physical adsorption to occur. Immobilization of a polypeptide by chemical bonding typically involves the attachment of the polypeptide to the support material via a covalent bond.
In some embodiments, the dsRNA ligase polypeptide is immobilized via a spacer positioned between the dsRNA ligase polypeptide and the solid material. In some embodiments, the spacer is a peptide (e.g. a peptide comprising 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 75 or more, or 100 or more amino acids).
In some embodiments, the engineered dsRNA ligase polypeptide is immobilized using affinity immobilization. In some embodiments, the engineered dsRNA ligase polypeptide is immobilized using metal affinity immobilization, e.g. by contacting His- tagged engineered dsRNA ligase polypeptide with immobilized metal such as nickel, zinc, cobalt, or copper.
In some embodiments, the solid support material comprises a membrane, resin, solid carrier, or other solid phase material. A solid support material can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polymethacrylate, and polyacrylamide, as well as co-polymers and grafts thereof. A solid support material can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse phase silica or metal, such as gold or platinum. The configuration of a solid support material can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non -planar. Solid support materials can be porous or non-porous and can have swelling or non-swelling characteristics. A solid support material can be configured in the form of a well, depression, or other container, vessel, feature, or location. Solid support materials useful for immobilizing the dsRNA ligase polypeptide for carrying out a ligase reaction include but are not limited to beads or resins such as polymethacrylate, e.g., polymethacrylates with epoxy functional groups, polymethacrylates with amino epoxy functional groups, polymethacrylates, styrene/DVB copolymer or polymethacrylates with octadecyl functional groups.
Exemplary solid supports include, but are not limited to, chitosan beads, Eupergit C, IB-150, IB-350, IB-C435, IB-A369, IB-A161, IB-A171, IBS500, IB-S861, SEPABEADS (Mitsubishi), e.g., Sepabeads EC-EP, Sepabeads EC-HFA, Sepabeads EC-HG, Sepabeads EC-BU, Sepabeads EC-OD, Sepabeads EC-CM, Sepabeads EC-IDA, Sepabeads EC-EA, Sepabeads EC-HA, Sepabeads EC-QA, Sepabeads EXE, Sepabeads EXA, Dilbeads-TA, Amberzyme Oxirane, Amberlite XAD-7HP, Amberlite FPA98C1, Amberlite IRA958C1, Amberlite IRA67, Amberlite FPA90C1, Amberlite FPA40C1, Amberlite XAD18, Accurel EP100, ECR8206F/5730, ECR8206/5803, ECR8206M/5749, ReliZyme EP403, ReliZymeEPl 13, Lewatit VP OC 1600, Diaion WA20, Diaion WA21J, Diaion WA30, Dowex 66, Diaion HPA-25L, Lewatit VP OC 1064 MD PH, Lewatit VP OC 1163, Lifetech ECR8304F. Lifetech ECR8309F, Lifetech ECR8315F, Lifetech ECR8204F, Lifetech ECR8285, Lifetech ECR1090M, Lifetech ECR1030M, Lifetech ECR8806M, Chromalite (MAM2/F) D6591, Chromalite MIDA/M, Chromalite MIDA/M/Le, Chromalite MIDA/M/Co, Chromalite MIDA/M/Ni, Chromalite MIDA/M/Cu and Chromalite MIDA/M/Zn.
Polynucleotides, control sequences, expression vectors and host cells that can be used to produce engineered dsRNA ligase polypeptides
In another aspect, this disclosure provides polynucleotides encoding engineered polypeptides having dsRNA ligase activity described herein. The polynucleotides can be linked to one or more heterologous regulatory sequences that control gene expression to produce recombinant polynucleotides that are capable of expressing the engineered polypeptides. Expression constructs comprising a heterologous polynucleotide encoding an engineered dsRNA ligase may be introduced into a suitable host cell to express the corresponding engineered dsRNA ligase polypeptide.
As apparent to one skilled in the art, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide an illustration of all possible polynucleotides that encode the protein sequence of interest. The degeneracy of the genetic code, in which the same amino acid is encoded by selectable or synonymous codons, allows for the production of an extremely large number of polynucleotides, all of which encode the engineered dsRNA ligase polypeptides disclosed herein. Thus, upon determination of a particular amino acid sequence, one skilled in the art can generate any number of different polynucleotides by modifying one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, this disclosure specifically contemplates each and every possible alteration of a polynucleotide that can be made by selecting a combination based on possible codon selections, for any of the polypeptides disclosed herein, comprising those amino acid sequences of exemplary engineered polypeptides listed in Examples 7 to 12, any of the polypeptides disclosed as even sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668, and any of the polypeptides disclosed as even sequence identifiers of SEQ ID NOs: 4 to 300 and 602 to 634.
In various embodiments, the codons are preferably selected to accommodate the host cell in which the recombinant protein is produced. For example, codons preferred for bacteria are used to express genes in bacteria; codons preferred for yeast are used to express genes in yeast; and codons preferred for mammals are used for gene expression in mammalian cells.
In some embodiments, the disclosure provides a polynucleotide encoding an engineered dsRNA ligase polypeptide described above.
In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to a reference sequence that is an even numbered sequence identifier of SEQ ID NOs: 304 to 600 or 636 to 668, wherein the polypeptide has dsRNA ligase activity and exhibits higher enzyme activity than a polypeptide comprising the amino acid of SEQ ID NO: 2 and/or 302.
In some embodiments, the polynucleotide encodes an engineered dsRNA ligase polypeptide described herein and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference polynucleotide selected from the sequences having an odd numbered sequence identifier of SEQ ID NOs: 303 to 599 or 635 to 667, wherein: (i) the polynucleotide does not comprise SEQ ID NO: 301; and (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ ID NO: 302.
In some embodiments, the polynucleotide encodes an engineered dsRNA ligase polypeptide described herein and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference polynucleotide selected from the sequences having an odd numbered sequence identifier of SEQ ID NOs: 3 to 299 or 601 to 633, wherein: (i) the polynucleotide does not comprise SEQ ID NO: 1; and (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ ID NO: 2. It will be readily understood that polynucleotides having an odd numbered sequence identifier of SEQ ID NOs: 3 to 299 or 601 to 633 encode an engineered dsRNA ligase polypeptide comprising an N-terminal purification tag (SEQ ID NO: 669).
The isolated polynucleotides encoding engineered dsRNA ligase polypeptides can be manipulated to enable the expression of the engineered polypeptides in a variety of ways, which may comprise further modification of the sequences by codon optimization to improve expression, insertion into suitable expression elements with or without additional control sequences, and transformation into a host cell suitable for expression and production of the engineered polypeptides.
Depending on the expression vector, manipulation of the isolated polynucleotide prior to insertion of the isolated polynucleotide into the vector may be desirable or necessary. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided below: Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. Eds., Greene Pub. Associates, 1998, 2010 Year update.
The disclosure also provides an expression vector comprising the polynucleotide described herein. In some embodiments, the vector is selected from a plasmid, a cosmid, a bacteriophage, or a viral vector. Recombinant expression vectors typically comprise one or more expression regulatory regions, such as promoters and terminators, origin of replication and the like.
Polynucleotides encoding an engineered dsRNA ligase polypeptide described herein can be expressed by inserting the polynucleotide or the nucleic acid construct comprising the polynucleotide sequence into an appropriate expression vector. In generating the expression vector, the coding sequence is located in the vector such that the coding sequence is linked to a suitable control sequence for expression. The recombinant expression vector can be any vector (e.g. , a plasmid or virus) that can be conveniently used in recombinant DNA procedures and can result in the expression of a polynucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell to be introduced into. The vector can be linear or closed circular plasmid. The expression vector may be an autonomously replicating vector, i. e. , a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any tools for ensuring self-copying. Alternatively, the vector may be a vector that, when introduced into a host cell, integrates into the genome and replicates with the chromosome into which it is integrated. Moreover, a single vector or plasmid or two or more vectors or plasmids that together comprise the total DNA to be introduced into the genome of the host cell may be used.
Many expression vectors useful to the embodiments of the present disclosure are commercially available. An exemplary expression vector can be prepared by inserting a polynucleotide encoding an engineered dsRNA ligase polypeptide to plasmid pACYC-Duet-1 (Novagen), pBR322 Vector (New England Biolabs), pUC19 Vector (New England Biolabs) or pET T7 Expression Vectors (Novagen).
The disclosure also provides a host cell capable of expressing an engineered dsRNA ligase polypeptide described herein. In some embodiments, the host cell comprises the nucleic acid molecule described herein, or the vector described herein. In some embodiments, the host cell is Escherichia coli.
In some embodiments, the polynucleotide encoding the polypeptide is linked to one or more control sequences for expression of polypeptides in the host cell. Host cells for expression of polypeptides encoded by the expression vectors of the present disclosure are well known in the art, including, but not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium,' fungal cells (e.g., Saccharomyces cerevisiae or Pichia pastoris).' insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. An exemplary host cell is E. coli BL21 (DE3). The host cell may be wild-type or may be engineered through genomic editing. Suitable media and growth conditions for the above host cells are well known in the art.
Polynucleotides or vectors used to express polypeptides can be introduced into cells by a variety of methods known in the art. Techniques comprise, among others, electroporation, bio-particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Different methods of introducing polynucleotides into cells are known to those skilled in the art.
The host cell may be used to express and isolate the polypeptide described herein.
Process of producing an engineered dsRNA ligase polypeptide
Engineered dsRNA ligase can be obtained by subjecting a polynucleotide encoding an dsRNA ligase to mutagenesis and/or directed evolution. An exemplary directional evolution technique can be found in "Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing" (2009 John Wiley &Sons Asia (Pte) Ltd. ISBN: 978-0- 470-82314-9).
When the sequence of an engineered polypeptide is known, the encoding polynucleotide may be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present disclosure can be prepared by chemical synthesis using, for example, the classic phosphoramidite methods described by Beaucage et al., 1981, Tet Lett 22: 1859-69, or Matthes et al. People, 1984, EMBO J. 3: 801-05, as typically practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated, and cloned into a suitable vector, for example, in an automated DNA synthesizer. In addition, essentially any nucleic acid is available from any of a variety of commercial sources.
The disclosure provides a method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing a host cell described herein and obtaining an engineered dsRNA ligase polypeptide from the culture. In some embodiments, the process of preparing a polypeptide further comprises isolating the polypeptide. Engineered polypeptides may be expressed in suitable cells and isolated (or recovered) from the host cell and/or culture medium using any one or more of the well-known techniques for protein purification, the techniques for protein purification include, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifiigation and chromatography.
The invention also provides an engineered dsRNA ligase catalyst obtainable by culturing a host cell described herein, or from the method of preparing an engineered dsRNA ligase polypeptide described herein, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cells, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cells, an extract thereof, or isolated product of the extract.
Ligation reactions
The disclosure provides a method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting: (i) two or more oligonucleotide fragments; (ii) an engineered dsRNA ligase polypeptide disclosed herein; (iii) a source of ATP; and (iv) a divalent cation; to obtain an oligonucleotide.
Oligonucleotide products and fragments
Methods of the invention produce oligonucleotides by ligating two or more oligonucleotide fragments. The produced oligonucleotides (also referred to herein as “oligonucleotide products”) are nucleic acids which typically comprise up to 100 nucleotides. It will be understood that oligonucleotides described herein comprise RNA. It will also be understood that oligonucleotides described herein comprise a double-stranded region. As used herein, “oligonucleotide fragment” refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide product. Each oligonucleotide fragment corresponds to a portion of the oligonucleotide product.
In some embodiments, the oligonucleotide is a therapeutic oligonucleotide. In some embodiments, the therapeutic oligonucleotide is a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO). In some embodiments, the oligonucleotide is an aptamer.
In some embodiments, the oligonucleotide comprises an overhang. In some embodiments, the oligonucleotide comprises a 3’ overhang. In some embodiments, the oligonucleotide comprises a 5’ overhang. In some embodiments, the overhang comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. In some embodiments, the oligonucleotide comprises a blunt end. In some embodiments, the oligonucleotide comprises two blunt ends.
In some embodiments, the oligonucleotide is up to 20 nucleotides in length. In some embodiments, the oligonucleotide is up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, or up to 100 nucleotides in length. In some embodiments, the oligonucleotide is up to 60 nucleotides in length.
In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In some embodiments, the oligonucleotide is at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or 100 nucleotides in length.
In some embodiments, the oligonucleotide is 10-100 nucleotides in length. In some embodiments, the oligonucleotide is 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, or 15-25 nucleotides in length. In some embodiments, the oligonucleotide is 15-25 nucleotides in length.
As used herein, the two or more oligonucleotide fragments comprise one or more 3’ oligonucleotide fragments and one or more 5 ’ oligonucleotide fragments, wherein each of the one or more 3’ oligonucleotide fragments comprise a 5 ’-phosphate group and each of the one or more 5’ oligonucleotide fragments typically comprise a 3’ terminal ribonucleotide having a 3 ’-hydroxyl group.
In some embodiments, one or more of the oligonucleotide fragments comprises one or more mismatches. In some embodiments, one or more of the oligonucleotide fragments comprise an overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 3’ overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 5’ overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 3’ overhang and a 5’ overhang. In some embodiments, the overhang comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides.
In some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having an overhang that is complementary to the overhang of a second oligonucleotide fragment. In some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having a 3 ’ overhang and a 5 ’ overhang, wherein the 3’ overhang is complementary to the 5’ overhang of a second oligonucleotide fragment and the 5’ overhang is complementary to the 3’ overhang of a third oligonucleotide.
In some embodiments, one or more of the oligonucleotide fragments comprise a blunt end. In some embodiments, one or more of the oligonucleotide fragments comprise a 3’ overhang and a 5’ blunt end. In some embodiments, one or more of the oligonucleotide fragments comprise a 5’ overhang and a 3’ blunt end. In some embodiments, the 5’ terminal oligonucleotide fragment comprises a 3’ overhang and a 5’ blunt end. In some embodiments, the 3’ terminal oligonucleotide fragment comprise a 5’ overhang and a 3’ blunt end.
In some embodiments, two or more of the oligonucleotide fragments comprise two or more RNA oligonucleotide fragments. In some embodiments, the two or more RNA oligonucleotide fragments comprise double-stranded RNA (dsRNA) oligonucleotide fragments.
In some embodiments, one or more of the oligonucleotide fragments comprise DNA and RNA. For example, a portion of the oligonucleotide fragment may be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.
In some embodiments, one or more of the oligonucleotide fragments comprise one or two strands which are RNA, or a mixture of RNA, DNA, LNA, Morpholino, UNA (unlocked nucleic acid), TNA (threose nucleic acid), GNA (glycol nucleic acid), and/or FANA (Fluoroarabino nucleic acid), modified RNA, etc. As a non-limiting example, one or both strand(s) could be, for example, RNA except that one or more nucleotide(s) is replaced by DNA, LNA, Morpholino, UNA, TNA, GNA, and/or FANA, and/or modified RNA (e.g., any modified RNA disclosed herein or known in the art, such as 2’ modified RNA, including but not limited to 2’-F, 2’-0Me, 2’-0-M0E RNA, etc.).
In some embodiments, the two or more oligonucleotide fragments are the same length. In some embodiments, the two or more oligonucleotide fragments are different lengths. In some embodiments, each of the two or more oligonucleotide fragments are 3-20 nucleotides in length. In some embodiments, each of the two or more oligonucleotide fragments are 4-16 nucleotides in length. In some embodiments, each of the two or more oligonucleotide fragments are 4-16, 4-15, 5-15, 6-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 5-9, or 6-9 nucleotides in length.
In some embodiments, each of the two or more oligonucleotide fragments are at least 3 nucleotides in length. In some embodiments, each of the two or more oligonucleotide fragments are at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.
In some embodiments, the two or more oligonucleotide fragments comprise 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more oligonucleotide fragments.
In some embodiments, one or more ligation reactions are required to generate the oligonucleotide product. In some embodiments, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more ligation reactions are required to generate the oligonucleotide product.
In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises a chemical modification. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one modified backbone modification. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one modified nucleotide modification. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one sugar modification (e.g. at the 2’-position or 4’-position). In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises: (i) at least one modified backbone modification; (ii) and at least one modified nucleotide modification; and/or (iii) at least one sugar modification.
In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprise a modification selected from the group consisting of: 2'-O-methyl (2’-OMe), 2'-flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'- O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'- O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g. mesyl phosphoramidate), 2',3'-seco nucleotide mimic, 2'-F-arabino nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, vinylpho sphonate (e.g. 5’ vinylphosphonate), and cyclopropyl phosphonate deoxyribonucleotide. In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises a 2'-modification selected from the group consisting of: 2’-0Me, 2’-F, and 2'-deoxy.
In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide comprises at least one phosphorothioate or methylphosphonate intemucleotide linkage. In some embodiments, the oligonucleotide comprises at least one chiral phosphorothioate linkage.
In some embodiments, one or more of the oligonucleotide fragments and/or the oligonucleotide is conjugated to at least one ligand. The ligand may be conjugated to the sense strand, antisense strand or both strands, in any configuration e.g. at the 3 ’-end, 5 ’-end, non-end or a combination.
In some embodiments, the ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives. GalNAc is an amino sugar derivative of galactose which may be used as a targeting ligand in oligonucleotides intended for targeting to the liver, where it binds to the asialoglycoprotein receptors on hepatocytes. In some embodiments, the ligand comprises one or more GalNAc derivatives conjugated through a bivalent or trivalent branched carrier. In some embodiments, the ligand is a peptide or a peptidomimetic.
In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3’ end of the sense strand. In some embodiments, the ligand is conjugated to the 5’ end of the sense strand. In some embodiments, the ligand is conjugated to a non-end of the sense strand.
In some embodiments, the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the 3’ end of the antisense strand. In some embodiments, the ligand is conjugated to a non-end of the antisense strand.
In some embodiments, the oligonucleotide is an RNAi agent comprising at least one 2’-modified nucleotide selected from a group consisting of 2’-0Me, 2’-F, 2'-deoxy, 2'-deoxy- 2’-fluoro, and 2'-0-M0E. In some embodiments, the oligonucleotide is an RNAi agent wherein the sense strand is conjugated to one or more GalNAc ligand(s). In some embodiments, one or more of the oligonucleotide fragments comprises at least one 2’- modified nucleotide selected from a group consisting of 2’-0Me, 2’-F, 2'-deoxy, 2'-deoxy-2’- fluoro, and 2'-0-M0E. In some embodiments, one or more of the oligonucleotide fragments is a dsRNA wherein the sense strand is conjugated to one or more GalNAc ligand(s).
In some embodiments, the method is performed with an oligonucleotide fragment concentration of at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM. In some embodiments, the method is performed with at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM of each oligonucleotide fragment. In some embodiments, the method is performed with equimolar amounts of each of the two or more oligonucleotide fragments.
In some embodiments, the method produces at least 15 g of oligonucleotide product per litre of reaction mixture. In some embodiments, the method produces at least 16 g, at least 17 g, at least 18 g, at least 19 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90, or at least 100 g of oligonucleotide product per litre of reaction mixture.
Engineered dsRNA ligase polypeptide
The method is performed using an engineered dsRNA ligase as described herein.
In some embodiments, the method is performed using about 1 g/L engineered dsRNA ligase polypeptide, optionally 1.1 g/L, 1.15 g/L, 1.2 g/L, 1.25 g/L, 1.3 g/L, 1.35 g/L, 1.4 g/L, 1.45 g/L, 1.5 g/L, 1.55 g/L, 1.6 g/L, 1.65 g/L, 1.7 g/L, 1.75 g/L, 1.8 g/L, 1.85 g/L, 1.9 g/L, 1.95 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.25 g/L, 3.5 g/L, 3.75 g/L, 4 g/L, 4.5 g/L or 5 g/L engineered dsRNA ligase polypeptide.
Source of ATP
The enzymatic activity of dsRNA ligase requires ATP as a cofactor. One molecule of ATP is converted to AMP per ligation reaction. The catalytic mechanism of dsRNA ligase and the role of ATP in nucleic acid ligation reactions are described above.
In some embodiments, the source of ATP is ATP. In some embodiments, the method is performed using a stoichiometric concentration of ATP. In some embodiments, the method is performed using a stoichiometric excess of ATP. The skilled person can readily determine the stoichiometric concentration of ATP required for a given ligation based on the concentration of the oligonucleotide fragments and the number of ligation reactions required to produce the oligonucleotide product.
In some embodiments, the method is performed using an ATP and/or AMP concentration of about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28mM or about 30mM.
In some embodiments, the source of ATP is an ATP regeneration system. In some embodiments, the ATP regeneration system comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and/or ATP. Advantageously, the use of an ATP regeneration system overcomes the requirement for high concentrations of ATP to achieve complete ligation. The ATP regeneration system described herein comprises PPK and polyphosphate. PPK generates ATP from AMP using polyphosphate as a phosphate donor. ATP that is converted to AMP during the ligation reaction can be regenerated to ATP by PPK and used as a cofactor in a subsequent ligation reaction. This cycling of ATP obviates the need for high ATP concentration in the starting reaction. Instead, the reaction can be performed using sub- stoichiometric concentrations of ATP, and/or using the cheaper alternative, AMP.
“Polyphosphate kinases” or “PPKs” are a family of enzymes which catalyze the formation of ATP from AMP and polyphosphate.
In some embodiments, the PPK is PPK12. In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 670, which is the amino acid sequence of PPK12. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 670.
In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 671, which is the amino acid sequence of an optimized PPK12. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 671.
In some embodiments, the PPK is Acinetobacter johnsonii polyphosphate: AMP phosphotransferase (AjPAP) (UniProt ID: Q83XD3). In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 672, which is the amino acid sequence of AjPAP. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 672.
In some embodiments, the PPK is used in the form of whole cell, crude extract (e.g. cell free lyophilized extract or cell lysates), isolated polypeptide, or purified polypeptide. In some embodiments, the PPK polypeptide is used in an immobilized form as described herein, such as immobilized on a resin.
In some embodiments, the method is performed using about 1 g/L PPK, optionally 1.1 g/L, 1.15 g/L, 1.2 g/L, 1.25 g/L, 1.3 g/L, 1.35 g/L, 1.4 g/L, 1.45 g/L, 1.5 g/L, 1.55 g/L, 1.6 g/L, 1.65 g/L, 1.7 g/L, 1.75 g/L, 1.8 g/L, 1.85 g/L, 1.9 g/L, 1.95 g/L, 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, 3.25 g/L, 3.5 g/L, 3.75 g/L, 4 g/L, 4.5 g/L or 5 g/L PPK.
In some embodiments, the polyphosphate is a polyphosphate salt. In some embodiments, the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
In some embodiments, the method is performed using a stoichiometric excess of polyphosphate. In some embodiments, the method is performed using a polyphosphate concentration of at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.
In some embodiments, wherein the method is performed in the presence of PPK and polyphosphate, the method is performed in the presence of AMP. In some embodiments, wherein the method is performed in the presence of PPK and polyphosphate, the method is performed using a sub-stoichiometric concentration of ATP and/or AMP.
Divalent cation
The enzymatic activity of dsRNA ligases requires the presence of a divalent cation. The enzymatic activity of PPKs requires the presence of a divalent cation. In some embodiments, the divalent cation comprises Mg2+ and/or Mn2+.
In some embodiments, the method is performed with a divalent cation concentration of 5-100 mM, 10-100 mM, 15-100 mM, 20-100 mM, 30-100 mM, 5-90 mM, 5-80 mM, 5-70 mM, 5-60 mM, 5-50 mM, or 30-50 mM. In some embodiments, the method is performed with a divalent cation concentration of at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.
In some embodiments, the method further comprises purifying the oligonucleotide product from the reaction mixture. In some embodiments, the oligonucleotide product is at least 80% pure, optionally wherein the oligonucleotide product is at least 85%, at least 90%, at least 95% pure, optionally wherein the oligonucleotide product is at least 98% pure, optionally wherein the oligonucleotide product is at least 99% pure, optionally wherein the oligonucleotide product is at least 99.5% pure, optionally wherein the oligonucleotide product is at least 99.9% pure. An oligonucleotide product that is pure does not contain oligonucleotide fragments, intermediate ligation products, or side products arising from nonspecific ligation. The oligonucleotide product may be purified or isolated using any method known in the art, for example using gel extractions or using cellulose-based matrices.
The disclosure also provides an oligonucleotide produced by a method described herein. The oligonucleotide may be in any suitable buffer solution. In some embodiments, the buffer solution is selected from Tris buffer (e.g. Tris-HCl), phosphate buffer, HEPES, MOPS (3-(A-morpholino)propancsulfonic acid), and triethanolamine (TEOA) buffer. In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer solution further comprises an agent for controlling the osmolarity of the solution, such that the osmolarity is kept at a desired value, e.g., at the physiologic values of the human plasma. Solutes which can be added to the buffer solution to control the osmolarity include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolarity of the solution is a salt. In some embodiments, the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride.
Reaction conditions
As disclosed herein and exemplified in the examples, the present disclosure contemplates a range of suitable reaction conditions that may be used in the methods described herein, including but not limited to pH, temperature, buffers, substrate loadings, enzyme loading, cofactor loading, pressure, and reaction time. Additional suitable reaction conditions for ligation reactions described herein can be readily optimized by routine experimentation, e.g. performing the method described herein under experimental reaction conditions of varying reagent concentration, pH, temperature, and detecting the rate of oligonucleotide product formation.
In any of the embodiments of the process disclosed herein, the reaction conditions may include a suitable pH. As noted above, the desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer, or a combination of buffer and added acid or base. The pH of the reaction mixture can be controlled before and/or during the reaction. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 8, a pH of about 5 to about 8, a pH of about 6 to about 8, or a pH of about 7 to about 8. In some embodiments, the reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8.
In any of the embodiments of the method disclosed herein, suitable temperatures can be used for the reaction conditions, taking into consideration of, for example, the increase in reaction rate at higher temperatures, the activity of the enzyme for sufficient duration of the reaction. Accordingly, in some embodiments, suitable reaction conditions include a temperature of about 10°C to about 60°C, about 10°C to about 50°C, about 25 °C to about 50°C, about 25°C to about 40°C, about 25°C to about 30°C, or about 10°C to about 30°C. In some embodiments, suitable reaction temperatures include a temperature of about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a certain temperature throughout the reaction. In some embodiments, the temperature during the enzymatic reaction may be adjusted over a temperature profile during the course of the reaction.
The reaction may be performed in any suitable buffer solution. In some embodiments, the buffer solution is selected from Tris buffer (e.g. Tris-HCl), phosphate buffer, HEPES, MOPS (3-(A-morpholino)propanesulfonic acid), and triethanolamine (TEOA) buffer. In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline (PBS).
In some embodiments, the reaction mixture further comprises a reducing agent, optionally DTT (Dithiothreitol).
In carrying out the ligation reactions described herein, the engineered dsRNA ligase polypeptide may be added to the reaction mixture in different formulation forms, as frozen or lyophilized whole cells (FWC or LWC) transformed with the gene encoding the engineered dsRNA ligase polypeptide and/or as cell lysate or lyophilized cell lysate of such cells, so called shake flask powder (SFP), where the cell debris was removed and/or further purified as fermentation powder (FP). Whole cells transformed with gene(s) encoding the engineered dsRNA ligase polypeptide or cell extracts, lysates thereof, and isolated enzymes can be used in a wide variety of different forms, including solids (e.g., lyophilized, spray dried, or the like) or semisolid (e.g., a crude paste). The cell extract or cell lysate may be partially purified by precipitation (e.g. , ammonium sulfate, polyethyleneimine, heat treatment or the like), followed by desalting procedures (e.g., ultrafiltration, dialysis, and the like) prior to lyophilization. Any of the enzyme preparations can be immobilized to a solid phase material (such as a resin).
In any of the embodiments of the process disclosed herein, wherein an engineered polypeptide is expressed in the form of a secreted polypeptide, a culture medium containing the secreted polypeptide can be used in the process herein.
In any of the embodiments of the process disclosed herein, the solid reactants (e.g. , enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray dried, etc.), solutions, emulsions, suspensions, and the like. The reactants can be readily lyophilized or spray-dried using methods and instrumentation known to one skilled in the art. For example, the protein solution can be frozen at -80 °C in small aliquots, and then added to the pre-chilled lyophilization chamber, followed by the application of a vacuum.
In any of the embodiments of the process disclosed herein, the order of addition of reactants is not critical. The reactants may be added together to the solvent at the same time or alternatively, some reactants may be added separately, and some may be added together at different time points.
The methods of performing a ligation reaction may comprise the further step of isolating the oligonucleotide product of the enzymatic reaction. In particular, this step is typically performed after completion of the enzymatic reaction. The oligonucleotide is in particular typically separated from one or more, in particular essentially all of the other components of the reaction mixture. For example, the oligonucleotide is typically separated from the remaining substrate, side products, and/or enzymes. Isolation of the oligonucleotide may be achieved by means and techniques known in the art, e.g. by separating oligonucleotides based on their size such as by gel electrophoresis and gel extractions or using cellulose-based matrices. In some embodiments, the method further comprises purifying the oligonucleotide by ultrafiltration and chromatography. Modifications
In some embodiments, the oligonucleotide fragment(s) and/or the oligonucleotide comprises a modification, e.g. a chemical modification. As used herein, the term “oligonucleotide fragment(s)” means one or more oligonucleotide fragments. It will be appreciated that modifications which are present in the oligonucleotide fragment(s) are typically present in the oligonucleotide produced from said oligonucleotide fragment(s). In some embodiments, modification(s) are introduced to and/or removed from the oligonucleotide product.
In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises a chemical modification. In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one backbone modification. In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one nucleotide modification. In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one sugar modification (e.g. at the 2’-position or 4’-position). In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises: (i) at least one backbone modification; (ii) at least one nucleotide modification; and/or (iii) at least one sugar modification.
Modifications include, but are not limited to, end modifications of the terminal oligonucleotide fragments, e.g., 5 ’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, inverted linkages, etc.); base modifications, e.g. , replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages.
In some embodiments, a terminal oligonucleotide fragment and/or oligonucleotide comprises a cap. The term "cap” and the like include a chemical moiety attached to the end of a double-stranded nucleotide duplex, but is used herein to exclude a chemical moiety that is a nucleotide or nucleoside. A “3’ cap” is attached at the 3’ end of a nucleotide or oligonucleotide and protects the molecule from degradation, e.g., from nucleases, such as those in blood serum or intestinal fluid. A non-nucleotidic 3’ cap is not a nucleotide and can replace a TT or UU dinucleotide at the end of a blunt-ended oligonucleotide. In some embodiments, non-nucleotidic 3’ end caps are as disclosed in, for example, WO 2005/021749 and WO 2007/128477; and U.S. Pat. No. 8,097,716; U.S. Pat. No. 8,084,600; and U.S. Pat. No. 8,344,128. A “5’ cap” is atached at the 5’ end of a nucleotide or oligonucleotide. A cap should not interfere (or unduly interfere) with oligonucleotide activity.
In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises one or more mismatches. A mismatch is defined herein as a difference between the base sequence or length when two sequences are maximally aligned and compared. In the context of double -stranded oligonucleotides (in which two sequences are aligned antiparallel to each other) a mismatch is defined as a position wherein the base of one sequence is not complementary to the base of the other sequence. Thus, a mismatch is counted, for example, if a position in the first sequence has a particular base (e.g., A), and the corresponding position in the second sequence has a base which is not complementary to said base in the first sequence (e.g., G), when the first and second sequences are aligned antiparallel to each other. Note, however, that on a given RNA strand, a U can be replaced by T (either as RNA or, preferably, DNA, e.g., 2 ’-deoxy-thymidine); the replacement of a U with a T is not a mismatch as used herein, as either U or T can pair with A on the opposite strand. An RNA oligonucleotide can thus comprise one or more DNA bases, e.g., T. No mismatch is counted between a DNA portion(s) of an RNAi agent and the corresponding target mRNA if basepairing occurs (e.g., between A, G, C, or T in the DNA portion, and the corresponding U, C, G, or A, respectively in the mRNA).
A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide, which comprises a phosphate-sugar backbone but no base). A singlestranded nick in either sequence (or in the sense or anti-sense strand) is not counted as mismatch. Thus, as a non-limiting example, no mismatch would be counted if one sequence (in the 5 ’->3’ orientation) comprises the sequence AG, but the complementary sequence (in the 3 ’->5’ orientation) comprises the sequence TC with a single-stranded nick between the T and the C. A nucleotide modification in the sugar or phosphate is also not considered a mismatch. Thus, if one sequence comprises a G, and the complementary sequence comprises a modified C (e.g., 2 ’-modification) at the same position, no mismatch would be counted.
Thus, no mismatches are counted if modifications are made to the sugar, phosphate, or backbone of the oligonucleotide without modifying the base. Thus, in the context of double-stranded RNAi, a strand having a given sequence as an RNA would have zero mismatches from its complement sequence as a PNA; or morpholino; or LNA; or TNA; or GNA; or FANA; or a mix or chimera of RNA and DNA, TNA, GNA, FANA, Morpholino, UNA, LNA, and/or PNA, etc. No mismatch would occur between a nucleotide which is T, and a nucleotide which is A with a 5’ modification and/or a 2 ’-modification. The key feature of a mismatch (base replacement) is that it would not be able to base-pair with the corresponding base on the opposite strand. In addition, terminal overhangs such as “UU” or “dTdT” are not counted when counting the number of mismatches. In such cases, a mismatch is defined as a position wherein the base of one sequence does not match the base of the other sequence.
It is noted that dTdT (2'-deoxy-thymidine-5 ’-phosphate and 2'-deoxy-thymidine-5’- phosphate), or in some cases, TT or UU, can be added as a terminal dinucleotide cap or extension to one or both 3 ’-ends of the oligonucleotide, but this cap or extension is not included in the calculation of the total number of mismatches and is not considered part of the target sequence. This is because the terminal dinucleotide protects the ends from nuclease degradation but does not contribute to target specificity (Elbashir et al. 2001 Nature 411: 494- 498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12: 163- 176).
There are several examples in the art describing sugar, base, phosphate and backbone modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base modifications. Sugar modification of nucleic acid molecules are extensively described in the art.
Additional modifications and conjugations of oligonucleotides have been described. Soutschek et al. 2004 Nature 432: 173-178 presented conjugation of cholesterol to the 3’-end of the sense strand of an siRNA molecule by means of a pyrrolidine linker, thereby generating a covalent and irreversible conjugate. Chemical modifications (including conjugation with other molecules) of oligonucleotides may also be made to improve the in vivo pharmacokinetic retention time and efficiency.
In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises a modified base. The disclosure encompasses an oligonucleotide and oligonucleotide fragments with a substitution of a single nucleotide at a given position with a modified version of the same nucleotide. Thus a nucleotide (A, G, C or U) can be replaced by a modified base selected from 5-fluorouracil, 5 -bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5 -(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2 -thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5 -methylcytosine, N6-adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5- methoxyaminomethyl-2 -thiouracil, beta-D-mannosylqueosine, 5'- methoxy carboxymethyluracil, 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil - 5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2 -thiouracil, 3-(3-amino-3-N-2 -carboxypropyl) uracil, 2,6-diaminopurine, 5 -hydroxymethyl cytosine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiothymine, 5-propynyl ( — C=C — CHs) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methyladenine, 2-F- adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3 -deazaadenine..
Additional modified variants include the addition of any other moiety (e.g., a radiolabel or other tag or conjugate) to the oligonucleotide or oligonucleotide fragment; provided that the base sequence is identical, the addition of other moieties produces a “modified variant” (with no mismatches).
In addition to these modifications and patterns (e.g., formats) for modifications, other modifications or sets of modifications of the sequences provided can be generated using common knowledge of nucleic acid modification. These various embodiments and embodiments of the oligonucleotides of the present disclosure can be used in RNA interference.
In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises a modification that causes the oligonucleotide to have increased stability in a biological sample or environment (e.g., cytoplasm, interstitial fluid, blood serum, lung or intestinal lavage).
In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises a modification that promotes cleavage by the RNA-induced silencing complex (z. e. a “RISC cleavage site”). The RISC cleavage site is the site on the target at which cleavage occurs. In some embodiments, the antisense strand comprises a RISC cleavage site. For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5’-end. As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13 of the antisense strand.
In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises a modified backbone. As used herein, an unmodified backbone consists of 3’ to 5’ phosphodiester bonds. A modified backbone may comprise non-natural intemucleoside linkages. Oligonucleotides having a modified backbone include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
Oligonucleotide fragments comprising a modified backbone include, but are not limited to, those that do not have a phosphorus atom in the backbone. Modified backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (e.g. 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (e.g. mesyl phosphoramidate, 3'-amino phosphoramidate and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5 '-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3’-5* to 5’-3* or 2'-5' to 5’-2*.
Oligonucleotide fragments comprising a modified backbone that does not include a phosphorus atom therein may have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises at least one phosphonate linkage, wherein the phosphonate is a modified phosphonate selected from the group consisting of: phosphorothioate (which may be an Rp isomer or an .S'p isomer):
Figure imgf000063_0001
methylphosphonate :
Figure imgf000064_0001
methoxypropylphosphonate :
Figure imgf000064_0002
5 ’ -methylphosphonate : phonate:
Figure imgf000065_0001
5’-phosphorothioate;
Figure imgf000065_0002
and peptide nucleic acid:
Figure imgf000065_0003
In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises: at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’-modified nucleotide; at least one 5’-uridine-guanine-3’ (5’-ug-3’) dinucleotide, wherein the 5 ’-uridine is a 2’-modified nucleotide; at least one 5’-cytidine-adenine-3’ (5’-ca-3’) dinucleotide, wherein the 5’-cytidine is a 2’-modified nucleotide; or at least one 5’-uridine- uridine-3’ (5’-uu-3’) dinucleotide, wherein the 5’-uridine is a 2’-modified nucleotide. These dinucleotide motifs are particularly prone to serum nuclease degradation (e.g. RNase A). Chemical modification at the 2'-position of the first pyrimidine nucleotide in the motif prevents or slows down such cleavage. This modification recipe is also known under the term 'endo light'.
In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprise a modified nucleobase, wherein the modified nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particular embodiment, the modified nucleobase is difluorotolyl. In some embodiments, wherein the oligonucleotide and/or oligonucleotide fragment(s) is double-stranded, only one of the two strands contains a modified nucleobase. In some embodiments, wherein the oligonucleotide and/or oligonucleotide fragment(s) is double-stranded, both of the strands contain a modified nucleobase.
In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises a modified sugar. Sugar modifications typically involve chemical modification of the sugar moiety of RNA or DNA. Sugar modifications include, but are not limited to, one of the following at the 2’-position: OH; F; O-, S-, orN-alkyl; O-, S-, orN-alkenyl; O-, S- or N- alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to Cio alkyl or C2 to Cio alkenyl and alkynyl. Exemplary modifications include O[(CH2)nO] mCHs, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Oligonucleotide fragments for use in the methods described herein may include one of the following at the 2’ position: Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a therapeutic RNA, or a group for improving the pharmacodynamic properties of a therapeutic RNA. In some embodiments, the modification comprises a 2 ’-methoxy ethoxy (also known as 2 ’-O-(2 -methoxyethyl) or 2’-O-MOE), 2’- dimethylaminooxyethoxy (also known as 2’-DMAOE), and 2 ’-dimethylaminoethoxy ethoxy (also known in the art as 2’-O-dimethylaminoethoxyethyl or 2’-DMAEOE). Further exemplary modifications include: 5’-Me-2’-F nucleotides, 5’-Me-2’-Ome nucleotides, 5’- Me -2 ’-deoxynucleotides, 2 ’-alkoxyalkyl; and 2’-NMA (N-methylacetamide). Other modifications include 2’-methoxy (2’-OCH3), 2 ’-aminopropoxy (2’- OCH2CH2CH2NH2) and 2 ’-fluoro (2’-F). Similar modifications can also be made at other positions on an RNA, particularly the 3 ’ position of the sugar on the 3 ’ terminal nucleotide or in 2’-5’ linked dsRNA and the 5’ position of 5’ terminal nucleotide.
In some embodiments, the oligonucleotide fragment(s) and/or oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modification is selected from the group consisting of: 2’-O-methyl (2’-0me), 2’-flouro (2’-F), 2’-deoxy, 2’- deoxy-2’ -fluoro, 2’-O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl (2’-O-AP), 2’-O- dimethylaminoethyl (2’-0-DMA0E), 2’-O-dimethylaminopropyl (2’-0-DMAP), 2 -0- dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’-O-N-methylacetamido (2’-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g. mesyl phosphoramidate), 2’, 3 ’-seco nucleotide mimic, 2’-F -arabino nucleotide, abasic nucleotide, 2’-amino modified nucleotide, 2 ’-alkyl -modified nucleotide, morpholino nucleotide, vinylphosphonate (e.g. 5’ vinylphosphonate), and cyclopropyl phosphonate deoxyribonucleotide. In some embodiments, one or more of the oligonucleotide fragments comprises a 2 ’-modification selected from the group consisting of: 2’-0me, 2’-F, and 2’- deoxy. In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises one or more 3'-O-methyl nucleotide.
In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises a 2'-modification selected from the group consisting of: 2'-O-methyl (2’-0Me), 2'- flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-0-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O- dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-0-DMAE0E), 2'- O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), phosphoramidate (e.g. mesyl phosphoramidate), 2',3'-seco nucleotide mimic, 2'-F-arabino nucleotide, abasic nucleotide, 2'- amino modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, vinylphosphonate (e.g. 5’ vinylphosphonate), deoxyribonucleotide, and cyclopropyl phosphonate. In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises one or more 3'-O-methyl nucleotide.
In some embodiments, the oligonucleotide and/or oligonucleotide fragment(s) comprises a bridged nucleic acid. In some embodiments, the bridged nucleic acid is locked nucleic acid. In some embodiments, the bridged nucleic acid is constrained ethyl bridged nucleic acid:
Figure imgf000068_0001
In some embodiments, all pyrimidines (uridine and cytidine) are 2’ O-methyl- modified nucleosides.
In some embodiments, the sense and/or antisense strand is conjugated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, modified or unmodified nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium- molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.
In some embodiments, the antisense strand comprises at least one 2’-0Me modified nucleotide. In some embodiments, the antisense strand comprises at least one 2’-F modified nucleotide. In some embodiments, the antisense strand comprises at least one 2’-deoxy modified nucleotide. In some embodiments, the antisense strand comprises at least one 2’- OMe modified nucleotide, at least one 2’-F modified nucleotide, or at least one 2’-deoxy modified nucleotide, or any combination thereof. In some embodiments, the antisense strand comprises alternating 2’-0Me and 2’-F modified nucleotides. In some embodiments, the antisense strand comprises at least one 5’ vinylphosphonate. In some embodiments, the antisense strand comprises at least one chiral phosphorothioate linkage. In some embodiments, the antisense strand comprises at least one GNA. In some embodiments, the sense strand comprises at least one 2’-0Me modified nucleotide. In some embodiments, the sense strand comprises at least one 2’-F modified nucleotide. In some embodiments, the sense strand comprises at least one 2’-deoxy modified nucleotide. In some embodiments, the sense strand comprises at least one 2’-0Me modified nucleotide, at least one 2’-F modified nucleotide, or at least one 2’ -deoxy modified nucleotide, or any combination thereof. In some embodiments, the sense strand comprises alternating 2’-0Me and 2’-F modified nucleotides. In some embodiments, the antisense strand and the sense strand each comprise at least one 2’-0Me modified nucleotide. In some embodiments, the antisense strand and the sense strand each comprise at least one 2’-F modified nucleotide. In some embodiments, the antisense strand and the sense strand each comprise alternating 2’-0Me and 2’-F modified nucleotides. In some embodiments, the sense strand comprises at least one 5’ vinylphosphonate. In some embodiments, the sense strand comprises at least one chiral phosphorothioate linkage. In some embodiments, the sense strand comprises at least one GNA.
In some embodiments, the sense strand comprises alternating 2’-0Me and 2’-F modified nucleotides over the full length of the sense strand. In some embodiments, the sense strand comprises alternating 2’-0Me and 2’-F modified nucleotides over part of the length of the sense strand e.g. over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides of the sense strand.
In some embodiments, the antisense strand comprises alternating 2’-OMe and 2’-F modified nucleotides over the full length of the antisense strand. In some embodiments, the antisense strand comprises alternating 2’-OMe and 2’-F modified nucleotides over part of the length of the antisense strand e.g. over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more nucleotides of the antisense strand.
In some embodiments, the sense strand and antisense strand each comprise alternating 2’-OMe and 2’-F modified nucleotides over the full length of the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand comprise alternating 2’-OMe and 2’-F modified nucleotides over part of the length of the sense strand and the antisense strand e.g. over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or more nucleotides of the sense strand and the antisense strand.
Ligands
In some embodiments, one or more of the oligonucleotide fragments is conjugated to at least one ligand. In some embodiments, the oligonucleotide product is conjugated to at least one ligand. The ligand may be conjugated to the sense strand, antisense strand or both strands, in any configuration e.g. at the 3 ’-end, 5 ’-end, non-end or a combination.
In some embodiments, the ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives. In some embodiments, the ligand comprises one or more GalNAc derivatives conjugated through a bivalent or trivalent branched carrier. In some embodiments, the ligand is:
Figure imgf000070_0001
In some embodiments, the ligand is:
Figure imgf000070_0002
In some embodiments, the ligand is:
Figure imgf000070_0003
In some embodiments, the ligand is:
Figure imgf000071_0001
In some embodiments, the ligand is:
Figure imgf000071_0002
In some embodiments, the ligand is:
Figure imgf000071_0003
In some embodiments, a ligand alters the distribution, targeting or lifetime of the molecule into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Ligands providing enhanced affinity for a selected target are also termed targeting ligands.
Some ligands can have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the oligonucleotide, or a composition comprising the oligonucleotide, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH- dependent membrane activity and fusogenicity. In some embodiments, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the oligonucleotide, or a composition comprising the oligonucleotide, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). The endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.
Ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligonucleotide.
Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polylysine (PLL), poly L aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Examples of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide .
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N- acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptidomimetic or an aptamer.
Other examples of ligands include dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelator (e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis- O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, 03- (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF -KB .
In some embodiments, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue.
In some embodiments, the ligand is a peptide or a peptidomimetic. A peptidomimetic is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phagedisplay library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991).
As used herein, a “peptide moiety” can range in length from about 5 amino acids to about 50 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. An arginine-glycine-aspartic acid (RGD)-peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an oligonucleotide to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. Peptides that target markers enriched in proliferating cells can be used. For example, RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin. Thus, the ligand may comprise RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, or synthetic RGD mimics.
Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, , or y peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides .
Ligands can be coupled to the oligonucleotide fragment(s) and/or oligonucleotide at various places, for example, 3 ’-end, 5 ’-end, and/or at an internal (“non-end”) position. In some embodiments, the ligand is attached via an intervening tether, e.g., a carrier described herein. The ligand or tethered ligand may be present on a monomer when the monomer is incorporated into the oligonucleotide fragment(s) and/or oligonucleotide. In some embodiments, the ligand may be incorporated via coupling to a “precursor” monomer after the “precursor” monomer has been incorporated into the oligonucleotide fragment and/or the oligonucleotide. For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH2)nNH2 may be incorporated into a growing oligonucleotide fragment. In a subsequent operation, i.e., after incorporation of the precursor monomer into the oligonucleotide fragment, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer’s tether.
In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated, e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the oligonucleotide fragment(s) and/or the oligonucleotide, a ligand having complementary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
In some embodiments, the ligand is conjugated to nucleobases, sugar moieties, or intemucleosidic linkages of the oligonucleotide fragment(s) and/or oligonucleotide. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6- positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The T position can also be attached to a conjugate moiety, such as in an abasic residue. Intemucleosidic linkages can also bear conjugate moieties. For phosphorus- containing linkages (e.g., phosphodiester, phosphorothioate (e.g. chrial phosphorothioate), phosphorodithiotate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing intemucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3’ end of the sense strand. In some embodiments, the ligand is conjugated to the 5’ end of the sense strand. In some embodiments, the ligand is conjugated to a non-end of the sense strand.
In some embodiments, the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the 3’ end of the antisense strand. In some embodiments, the ligand is conjugated to a non-end of the antisense strand.
The ligand may be attached via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a nucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
Wherein the oligonucleotide fragment is a dsRNA, the sense and/or antisense strand may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.
In some embodiments, one or more oligonucleotide fragments comprise the sequence “TT”, “dTdT”, “dTsdT” or “UU” as a single-stranded overhang at the 3’ end, also termed herein a terminal dinucleotide or 3’ terminal dinucleotide. dT is 2'-deoxy-thymidine-5’- phosphate and sdT is 2'-deoxy Thymidine 5'-phosphorothioate. Terminal dinucleotide “UU” is UU or 2’-0Me-U 2’-0Me-U, and the terminal TT and the terminal UU can be in the inverted/reverse orientation. The terminal dinucleotide (e.g., UU) is a modified variant of the dithymidine dinucleotide commonly placed as an overhang to protect the ends of siRNAs from nucleases (see, for example, Elbashir et al. 2001 Nature 411: 494-498; Elbashir et al. 2001 EMBO J. 20: 6877-6888; and Kraynack et al. 2006 RNA 12: 163-176). A terminal dinucleotide is known from these references to enhance nuclease resistance but not contribute to target recognition.
In some embodiments, one or both terminal oligonucleotide fragments comprise a 3 ’ end cap instead of or in addition to a terminal dinucleotide to stabilize the end from nuclease degradation provided that the 3’ end cap is able to both stabilize the oligonucleotide (e.g., against nucleases) and not interfere excessively with its desired activity.
Wherein the oligonucleotide fragment is a dsRNA, the sense and/or antisense strand may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.
Additional embodiments
Embodiment 1. An engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,
352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,
388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422,
424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458,
460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,
496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530,
532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566,
568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and
(b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
Embodiment 2. An engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,
352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386,
388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422,
424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458,
460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,
496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530,
532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566,
568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636,
638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein the engineered dsRNA ligase polypeptide:
(a) has dsRNA ligase activity; and
(b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
Embodiment 3. The engineered dsRNA ligase polypeptide of Embodiment 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592.
Embodiment 4. The engineered dsRNA ligase polypeptide of Embodiment 2, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592 and 666.
Embodiment 5. An engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302. Embodiment 6. The engineered dsRNA ligase polypeptide of Embodiment 5, wherein the ligation reaction conditions include about 1 pM to about 10 mM oligonucleotide fragment, a source of ATP, about 5 mM to about 100 mM divalent cation, and about 0.5 g/L to about 10 g/L engineered dsRNA ligase polypeptide, pH of about 4.0 to about 8.0, and temperature of about 10 °C to about 50 °C.
Embodiment 7. The engineered dsRNA ligase polypeptide of Embodiment 5 or 6, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, XI 14, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.
Embodiment 8. The engineered dsRNA ligase polypeptide of Embodiment 7, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, or R; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302.
Embodiment 9. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-8, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158,
X163, X173, X178, X185, X190, X196, X216, X218, X221, X228, X230, X232, X235,
X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269,
X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.
Embodiment 10. The engineered dsRNA ligase polypeptide of Embodiment 9, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302.
Embodiment 11. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-10, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
Embodiment 12. The engineered dsRNA ligase polypeptide of Embodiment 11, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
Embodiment 13. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-12, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.
Embodiment 14. The engineered dsRNA ligase polypeptide of Embodiment 13, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302.
Embodiment 15. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-14, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X36, X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.
Embodiment 16. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-15, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; and X221 is l. Embodiment 17. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-16, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X218, X221 and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.
Embodiment 18. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-17, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
Embodiment 19. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-18, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
Embodiment 20. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-19, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X19, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X19 is D; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
Embodiment 21. The engineered dsRNA ligase polypeptide of any one of Embodiments 5-20, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X 53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
Embodiment 22. The engineered dsRNA ligase polypeptide of any of Embodiments 1- 21, wherein the engineered dsRNA ligase polypeptide comprises a purification tag.
Embodiment 23. The engineered dsRNA ligase polypeptide of Embodiment 22, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,
250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,
286, 288, 290, 292, 294, 296, 298 and 300.
Embodiment 24. The engineered dsRNA ligase polypeptide of Embodiment 22, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248,
250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284,
286, 288, 290, 292, 294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620,
622, 624, 626, 628, 630, 632, and 634.
Embodiment 25. A polypeptide immobilized on a solid material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide according to any one of Embodiments 1-24.
Embodiment 26. A polynucleotide encoding the engineered dsRNA ligase polypeptide of any one of Embodiments 1-24.
Embodiment 27. The polynucleotide of Embodiment 26, wherein the polynucleotide sequence is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,
203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,
239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,
275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 303, 305, 307, 309, 311,
313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,
349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,
385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419,
421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455,
457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491,
493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527,
529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,
565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, or 599. Embodiment 28. The polynucleotide of Embodiment 26, wherein the polynucleotide sequence is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201,
203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,
239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,
275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 303, 305, 307, 309, 311,
313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347,
349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383,
385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419,
421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455,
457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491,
493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527,
529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563,
565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599,
601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635,
637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667.
Embodiment 29. An expression vector comprising the polynucleotide according to any one of Embodiments 26-28.
Embodiment 30. The expression vector of Embodiment 29, which comprises a plasmid, a cosmid, a bacteriophage or a viral vector.
Embodiment 31. A host cell comprising the polynucleotide of any one of Embodiments 26-28 or the expression vector of Embodiment 29 or 30, optionally wherein the host cell is E. coli.
Embodiment 32. A method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing the host cell according to Embodiment 31 and obtaining an engineered dsRNA ligase polypeptide from the culture. Embodiment 33. An engineered dsRNA ligase catalyst obtainable by culturing the host cells according to Embodiment 31, or according to the method of Embodiment 32, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cell, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cell, an extract thereof, or isolated product of the extract.
Embodiment 34. A method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting:
(i) two or more oligonucleotide fragments;
(ii) an engineered dsRNA ligase polypeptide according to any one of Embodiments 1- 24;
(iii) a source of ATP; and
(iv) a divalent cation; to obtain an oligonucleotide.
Embodiment 35. The method of Embodiment 34, wherein the source of ATP comprises ATP.
Embodiment 36. The method of Embodiment 34 or 35, wherein the source of ATP comprises:
(a) polyphosphate kinase (PPK);
(b) polyphosphate; and
(c) AMP and/or ATP.
Embodiment 37. The method of Embodiment 36, wherein the PPK is selected from PPK12 or ajPAP.
Embodiment 38. The method of any one of Embodiments 36 or 37, wherein the method is performed using a sub-stoichiometric concentration of AMP and/or ATP. Embodiment 39. The method of any one of Embodiments 36-38, wherein the polyphosphate is a polyphosphate salt.
Embodiment 40. The method of Embodiment 39, wherein the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt).
Embodiment 41. The method of any one of Embodiments 34-40, wherein the divalent cation cofactor is Mg2+ or Mn2+.
Embodiment 42. The method of any one of Embodiments 34-41, wherein the method is performed with a divalent cation concentration of 5-100 mM, optionally 30-50 mM.
Embodiment 43. The method of any one of Embodiments 34-42, further comprising a step of purifying the oligonucleotide.
Embodiment 44. Use of the engineered dsRNA ligase polypeptide according to any one of Embodiments 1-24 in the production of an oligonucleotide from two or more oligonucleotide fragments.
Embodiment 45. The method of any one of Embodiments 34-43 or the use of
Embodiment 44, wherein the oligonucleotide is up to 60 nucleotides in length.
Embodiment 46. The method of any one of Embodiments 34-43 or 45 or the use of Embodiment 44 or 45, wherein each of the oligonucleotide fragments are 4-16 nucleotides in length, optionally 6-9 nucleotides in length.
Embodiment 47. The method of Embodiment 34-43, 45 or 46 or the use of any one of Embodiments 44-46, wherein one or more of the oligonucleotide fragment(s) comprises one or two overhangs.
Embodiment 48. The method of any one of Embodiments 34-43 or 45-47 or the use of any one of Embodiments 44-47, wherein one or more of the oligonucleotide fragments comprises a chemical modification. Embodiment 49. The method or use of Embodiment 48, wherein the chemical modification is selected from:
(a) a modified backbone, optionally selected from a phosphorothioate (e.g. chiral phosphorothioate) or methylphosphonate intemucleotide linkage;
(b) a modified nucleotide, optionally selected from 2'-O-methyl (2’-0Me), 2'-flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-0-M0E), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O- dimethylaminopropyl (2'-0-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O- DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g. mesyl phosphoramidate), 2',3'-seco nucleotide mimic, 2'-F-arabino nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, vinylphosphonate (e.g. 5’ vinylphosphonate), and cyclopropyl phosphonate deoxyribonucleotide; and/or
(c) conjugation to a ligand, optionally wherein the ligand comprises one or more N- Acetylgalactosamine (GalNAc) derivatives.
Embodiment 50. A composition comprising: i. the engineered dsRNA ligase polypeptide according to any one of Embodiments 1- 24; ii. a source of ATP; and iii. a divalent cation.
Embodiment 51. The composition of Embodiment 50, further comprising two or more oligonucleotide fragments.
Embodiment 52. A kit comprising: i. the engineered dsRNA ligase polypeptide according to any one of Embodiments 1- 24; ii. a source of ATP; iii. a divalent cation; and iv. instructions for use in a method of producing an oligonucleotide from two or more oligonucleotide fragments. Embodiment 53. The composition of Embodiment 50 or 51 or the kit of Embodiment 52, wherein the source of ATP comprises ATP.
Embodiment 54. The composition of any one of Embodiments 50, 51 or 53 or the kit of Embodiment 52 or 53, wherein the source of ATP comprises:
(a) polyphosphate kinase (PPK);
(b) polyphosphate; and
(c) AMP and/or ATP.
Embodiment 55. The composition or kit of Embodiment 54, wherein the PPK is selected from PPK 12 or ajPAP.
Embodiment 56. The composition of any one of Embodiments 50, 51 or 53-55 or the kit of any one of Embodiments 52-55, wherein the polyphosphate is a polyphosphate salt.
Embodiment 57. The composition or kit of Embodiment 56, wherein the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt). Embodiment 58. The composition of any one of Embodiments 50, 51 or 53-57 or the kit of any one of Embodiments 52-57, wherein the divalent cation cofactor is Mg2+ or Mn2+.
Different features and embodiments of the present disclosure are exemplified in the following representative examples, which are intended to be illustrative and not restrictive.
EXAMPLES
The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.
In the Examples below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and mb (milliliter); cm (centimeters); mm (millimeters); um and pm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); rpm (rotations per minute); psi and PSI (pounds per square inch); °C (degrees Centigrade); RT and rt (room temperature); OD600 (Optical density at 600 nm), CAM and cam (chloramphenicol); DMSO (dimethylsulfoxide); FP (Fermentation powder); FWC (Frozen whole cells), LWC (Lyophilized whole cells), PMBS (polymyxin B sulfate); IPTG (isopropyl P-D-l -thiogalactopyranoside); LB (Lysogeny broth); TB (Terrific Broth; 12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSOi): TEoA (triethanolamine buffer), HEPES (HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (commonly used laboratory E. coli strain, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); FIOP (fold improvements over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Coming (Coming, Inc., Palo Alto, CA); Dow Coming (Dow Coming, Corp., Midland, MI); and Gene Oracle (Gene Oracle, Inc., Mountain View, CA).
The sequences of the oligonucleotides referred to in parentheses (e.g. “siRNA (1)” and “oligonucleotide (2)") throughout the Examples are provided in Table 1.
EXAMPLE 1
Preparation of isolated enzymes
Polynucleotides encoding the polypeptides having ligase activity, were cloned into the pCKl 10900 vector system (See e.g., US Pat. App. No. 2006/0195947A1 FIG. 3 which is hereby incorporated by reference in its entirety) and subsequently expressed in E. coli W311O 7n/A under the control of the lac promoter. The expression vector also contained the Pl 5a origin of replication and the chloramphenicol (CAM) resistance gene.
E. coli W311O 7IMA cells were transformed with the pCKl 10900 plasmid containing the ligase-encoding genes. Transformed cells were plated out on Lysogeny broth (LB) agar plates containing 1% glucose and 30 pg/mL CAM, and grown overnight at 37° C. Subsequently single colonies were inoculated into 25 mL of LB supplemented with 30 pg/mL CAM and 1% glucose in a 250 ml baffled shake flask. The culture was grown overnight (16-20 hours and optical density (ODeoo) >3.8) in an incubator at 37°C, with shaking at 250 rpm. A I L shake flask containing 250 mL of Terrific Broth (TB) media with 30 pg/mL CAM, was inoculated with 5 mL of the grown overnight culture. The 250 mL culture was incubated at 30°C, 250 rpm, for 3 - 3.5 hours until ODeoo reached 0.6-0.8. Expression of the ligase gene was induced by the addition of isopropyl-P-D-thiogalactoside (IPTG) to a final concentration of 1 mM, and growth was continued for an additional 18-20 hours. Cells were harvested by transferring the culture into a centrifuge bottle, which was then centrifuged at 7,000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the remaining cell pellet lysed. For lysis, the cell pellet was resuspended in 30 mb of 50 mM Tris-buffer at pH 7.5 and lysed using a LM20 MICROFLUIDIZER® processor system (Microfluidics). Cell debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. Ligase enzymes were then isolated from the clarified lysate using standard techniques known in the art, including immobilized metal affinity chromatography.
EXAMPLE 2
Identification of dsRNA ligase activity for the production of siRNA (1)
To identify an enzyme with dsRNA ligase activity for the production of siRNA (1) comprising of oligonucleotides (2) and (3) a collection of ligases were first screened for the production of a surrogate product, siRNA (4) comprising of oligonucleotides (3) and (5). Oligonucleotide (5) has the same sequence as oligonucleotide (2) but does not contain a 3’- GalNAc moiety. siRNAs (1) and (4) and oligonucleotides (2), (3) and (5) are depicted in Figure 1; and the sequences of oligonucleotides (2), (3) and (5) are provided in Table 1.
Screening of the isolated ligases was performed in 20 pL reaction volumes in PCR tubes, each tube containing 50 mM Tris-buffer pH 7.5, either 1 mM ATP or 1 mM NAD+, 10 mM MgC12, 5 mM DTT and 10 pM (each) of substrate oligonucleotides (6 - 11) with 50 % (v/v) isolated ligase enzyme. Reactions were incubated in a thermocycler at 16 °C for 2 h and analyzed using standard techniques known in the art, including electrophoresis. A ligase with SEQ ID NO: 2 exhibited the highest dsRNA ligase activity towards the formation of siRNA (4). The activity of SEQ ID NO: 2 for the production of siRNA (1) was subsequently confirmed using multiple enzyme preparations including isolated enzyme (example 1), clarified lysate (example 4) and shake flask powder (SFP; example 5). Table 1 Oligonucleotide sequences
Figure imgf000092_0001
EXAMPLE 3
Preparation of cell pellets for high throughput (HTP) screening
Single colonies were picked in a 96-well format and grown in 190 pL LB media containing 1% glucose and 30 pg/mL CAM, at 30°C, 200 rpm, and 85% humidity. Following overnight growth, 20 pL of the grown cultures were transferred into a deep well plate containing 380 pL of TB media with 30 pg/mL CAM. The cultures were grown at 30°C, 250 rpm, with 85% humidity for approximately 2.5 hours. When the ODeoo of the cultures reached 0.4-0.8, expression of the ligase gene was induced by the addition of IPTG to a final concentration of 1 mM. Following induction, growth continued for 18-20 hours at 30°C, 250 rpm with 85% humidity. Cells were harvested by centrifugation at 4,000 rpm and 4°C for 10 minutes; the supernatant was then discarded. The cell pellets were stored at -80°C until ready for use.
EXAMPLE 4
Lysis and preparation of clarified lysate
Prior to performing the assay, the cell pellets were thawed and resuspended in 300 pL of lysis buffer (containing 1 g/L lysozyme, 0.5 g/L PMBS and 0.1 pL/mL or 0.2U/ml of commercial DNAse (New England BioLabs, M0303L) in 50 mM Tris-buffer at pH 7.5. The plates were agitated with medium-speed shaking for 2.5 hours on a microtiter plate shaker at room temperature. The plates were then centrifuged at 4,000 rpm for 10 minutes at 4°C, and the clarified supernatants were used in the HTP assay reaction for activity determination as described in the following examples.
EXAMPLE 5
Preparation of shake flask powder (SFP) and fermentation powder (FP)
Shake-flask procedures can be used to generate engineered dsRNA ligase polypeptide shake-flask powders (SFP), which are useful for secondary screening assays and/or use in the biocatalytic processes described herein. Shake flask powder preparation of enzymes provides a more concentrated preparation of the engineered enzyme, as compared to the cell lysate used in HTP assays.
Clarified lysate produced according to example 1 was collected, frozen at -80°C, and then lyophilized, using standard methods known in the art. Lyophilization of frozen clarified lysate provides a dry SFP comprising crude wild-type or engineered dsRNA ligase polypeptide.
EXAMPLE 6
Analytical method for activity and selectivity evaluation
Activity improvements of the engineered dsRNA ligases were analyzed by High Pressure Liquid Chromatography (HPLC) using the methods described in Table 6-1 and 6-2. HPLC methods with UV-detection were developed to analyze the formation of product oligonucleotides (2) and (3). The analytical methods aim for the shortest run time enabling good resolution of the product oligonucleotides (2) and (3). Consequently, the six substrate (6- 7, 9-12) and the four intermediate (13-16) oligonucleotides could not all be well resolved from each other. However, it is possible to resolve the well-defined GalNAc -containing oligonucleotides, including the substrate oligonucleotide (12), a reaction intermediate oligonucleotide (14) and the product oligonucleotide (2). Therefore, a pseudo-% conversion can be calculated, denoted with arbitrary units (AU), which considers only these well resolved species according to the following equation:
Figure imgf000093_0001
Whereby 8(2), 8(12) and 8<i4) are the extinction coefficient of oligonucleotides (2), (12) and (14) respectively. Using such a calculation an AU = 1.0 would imply that no more GalNAc- containing substrate or intermediate oligonucleotides are present in the reaction and that they have all be converted to GalNAc -containing product (2). In reality, for samples where AU = 1.0 the only other peak present in the chromatogram corresponds with the product oligonucleotide (2), and no other intermediates or starting materials can be identified. Furthermore, the ratio of the product oligonucleotides (2) and (3) are consistent with that of the authentic standard of siRNA product (1). Taken together, it can be concluded that AU = 1.0 is an approximation that is essentially equivalent to 100 % conversion.
Table 6-1: HPUC method 1 used for activity determination.
Figure imgf000094_0001
HPLC method 2 (Table 6-2) was developed from HPLC method 1 (Table 6-1) to improve the separation between the product oligonucleotide (3) and the substrate oligonucleotide (12).
Table 6-2: HPLC method 1 used for activity determination.
Figure imgf000095_0001
The activity improvements of the engineered dsRNA ligases of Example 12 were also analyzed with RapidFire® Mass Spectrometry (RF-MS) using the method described in Table 6-3. RF-MS aims to reduce the analytical time compared to HPLC analysis. The selective detection of product oligonucleotides (2) and (3) is obtained by the specific masses of each product oligonucleotide analyzed under the multi-single ion monitoring (SIM) mode. Relative dsRNA ligase activity is determined by comparing the sum of the MS signal of the five specific masses (given in Table 6-3) corresponding with each product oligonucleotide (2) and (3).
Table 6-3: RF-MS method used for activity determination.
Figure imgf000096_0001
The methods provided herein find use in analyzing the variants produced using the present invention. However, it is not intended that present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of the variants provided herein and/or produced using the methods provided herein. EXAMPLE 7
Round 1 Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 2 for Improved Production of siRNA product (1)
The engineered polynucleotide (SEQ ID NO: 1) encoding the polypeptide with dsRNA ligase activity of SEQ ID NO: 2 was used to generate the engineered polypeptides of Table 7-
1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrate oligonucleotides (6-7, 9-12) as compared to the starting polypeptide. Some polypeptides displayed improved product formation of either oligonucleotide product (2) or (3), or both oligonucleotide products (2) and (3) compared to the starting polypeptide as noted in Table 7-1. The sequences of oligonucleotides (2), (3), (6), (7) and (9-12) are provided in Table 1.
The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO:
2, as described below together with the analytical method described in Table 6- 1. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 1. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotide products (2) and (3).
The enzyme assays were carried out in 96-well PCR plates, in 50 pL total reaction volume per well. The reactions contained 2.5 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 100 pM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.5, 1 mM ATP, 10 mM MgCh and 5 mM DTT. The reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 2 h.
After incubation the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate. The plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 2 pL aliquot of the supernatant was removed from each well and added to a shallow well 96-well plate containing 98 pL of 5 mM EDTA solution (pH 7.0). The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-1. Selected ligase variants showing greater product formation of oligonucleotides (2) and (3) relative to SEQ ID NO:2 are shown in Table 7-1.
Figure imgf000098_0001
Figure imgf000099_0001
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 4 to 106 comprise an even numbered sequence identifier of SEQ ID NOs: 304 to 406, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 4 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 304.
Throughout the Examples, the position of a given mutation is provided relative to SEQ ID NO: 2 which includes (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669 and (ii) the wild-type dsRNA ligase polypeptide of SEQ ID NO: 302. The position of a given mutation relative to SEQ ID NO: 302 (i.e. the wild-type dsRNA ligase polypeptide without the purification tag) can be obtained by subtracting the 14 amino acid N-terminal purification tag from the SEQ ID NOs described in the Examples. For example, position X251 of SEQ ID NO: 2 corresponds to position X237 of SEQ ID NO: 302. EXAMPLE 8
Round 2 Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 70 for Improved Production of siRNA product (1)
The polynucleotide from example 7 SEQ ID NO: 69 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 70 was used to generate the engineered polypeptides of Table 8-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide. Some polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 8-1. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 70, as described below together with the analytical method described in Table 6-1.
Directed evolution began with the polynucleotide set forth in SEQ ID NO: 69. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
The enzyme assays were carried out in 96-well PCR plates, in 50 pL total reaction volume per well. The reactions contained either 1.25 or 2.5 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 100 pM (each) substrate oligonucleotides (6-7, 9- 12), 50 mM Tris-buffer at pH 7.5, 1 mM ATP, 10 mM MgCh and 5 mM DTT. The reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 2 h.
After incubation the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate. The plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 2 pL aliquot of the supernatant was removed from each well and added to a shallow well 96-well plate containing 98 pL of 5 mM EDTA solution (pH 7.0). The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-1. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO:70 are shown in Table 8- 1.
Figure imgf000101_0001
Figure imgf000102_0001
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 108 to 216 comprise an even numbered sequence identifier of SEQ ID NOs: 408 to 516, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 108 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 408.
EXAMPLE 9 Round 3 Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 188 for Improved Production of siRNA product (1)
The polynucleotide from example 8 SEQ ID NO: 187 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 188 was used to generate the engineered polypeptides of Table 9-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide. Some polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 9- 1. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 188, as described below together with the analytical method described in Table 6-2.
Directed evolution began with the polynucleotide set forth in SEQ ID NO: 187. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
The enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well. The reactions contained 20 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 1 mM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.0, 10 mM ATP, 20 mM MgCh, 5 mM DTT and 10 % (v/v) DMSO. The reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 2 h.
After incubation the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate. The plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 450 pL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 50 pL of the diluted sample into a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0). The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 188 are shown in Table 9-1.
Figure imgf000103_0001
Figure imgf000104_0001
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 218 to 246 comprise an even numbered sequence identifier of SEQ ID NOs: 518 to 546, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 218 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 518.
EXAMPLE 10
Round 4 Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 226 for Improved Production of siRNA product (1)
The polynucleotide from example 9 SEQ ID NO: 225 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 226 was used to generate the engineered polypeptides of Table 10-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide. Some polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 10-1. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 226, as described below together with the analytical method described in Table 6-2.
Directed evolution began with the polynucleotide set forth in SEQ ID NO: 225. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
The enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well. The reactions contained 2.5 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 1 mM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.0, 10 mM ATP, 20 mM MgCh, 5 mM DTT and 10 % (v/v) DMSO. The reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
After incubation the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate. The plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 450 pL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 50 pL of the diluted sample into a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0). The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 226 are shown in Table 10- 1.
Figure imgf000105_0001
Figure imgf000106_0001
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 248 to 282 comprise an even numbered sequence identifier of SEQ ID NOs: 548 to 582, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 248 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 548.
EXAMPLE 11
Round 5 Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 278 for Improved Production of siRNA product (1)
The polynucleotide from example 10 SEQ ID NO: 277 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 278 was used to generate the engineered polypeptides of Table 11-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrates oligonucleotides (6-7, 9-12) as compared to the starting polypeptide. Some polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide are noted in Table 11-1. The engineered polypeptides, having the amino acid sequences of even-numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 278, as described below together with the analytical method described in Table 6-2. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 277. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
The enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well. The reactions contained 10 % (v/v) of undiluted dsRNA ligase lysate, prepared as described in Example 4, 5 mM (each) substrate oligonucleotides (6-7, 9-12), 50 mM Tris-buffer at pH 7.0, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO. The reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
After incubation the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate. The plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 50 pL of the diluted sample into a deep well 96-well plate containing 450 pL of 5 mM EDTA solution (pH 7.0). The samples were diluted a third time by transferring 160 pL of the diluted sample into a deep well 96-well plate containing 640 pL of 5 mM EDTA solution (pH 7.0). The samples were diluted a final time by transferring 75 pL of the diluted sample into a shallow well 96-well plate containing 75 pL of 5 mM EDTA solution (pH 7.0). The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 278 are shown in Table 11-1.
Figure imgf000107_0001
Figure imgf000108_0001
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 284 to 300 comprise an even numbered sequence identifier of SEQ ID NOs: 584 to 600, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 284 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 584.
EXAMPLE 12
Round 6 Evolution and Screening of Engineered Polypeptides Derived from SEQ ID NO: 288 for Improved Production of siRNA product (1) and improved thermal stability
The polynucleotide from example 11 SEQ ID NO: 287 encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO: 288 was used to generate the engineered polypeptides of Table 12-1. These polypeptides displayed improved dsRNA ligase activity under the desired conditions e.g., the improvement in the formation of either oligonucleotide products (2) or (3), or preferably both oligonucleotide products (2) and (3) that was produced in situ from the substrate oligonucleotides (6-7, 9-12) as compared to the starting polypeptide. Some polypeptides displayed improved product formation of both oligonucleotide products (2) and (3) compared to the starting polypeptide, as noted in Table 12- 1. Furthermore, some polypeptides displayed improved thermal stability, resulting in higher residual activity following incubation of the dsRNA ligase solution at 30 °C for 1 h prior to setting up the reaction (Table 12-2). The engineered polypeptides, having the amino acid sequences of even- numbered sequence identifiers were generated from the “backbone” amino acid sequence of SEQ ID NO: 288, as described below together with the analytical method described in Table 6-3. Directed evolution began with the polynucleotide set forth in SEQ ID NO: 287. Libraries of engineered polypeptides were generated using various well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assay and analysis methods, described below, that measured the polypeptides’ ability to produce oligonucleotides (2) and (3).
The enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well. The cells were lysed according to Example 4, however 100 mM MOPS-buffer at pH 7.2 was used in place of 50 mM Tris at pH 7.5. To provide a thermal challenge and to identify hits that were more thermostable, the cell lysates were either undiluted or diluted 1 : 1 in 100 mM MOPS buffer, pH 7.2 and incubated at 30 °C and 4 °C respectively for 1 h. The reactions contained a final dsRNA ligase concentration of 40 % (v/v) for lysate incubated at 30 °C and 20% (v/v) for lysate incubated at 4 °C. In addition, the ligation reactions contained 3 mM (each) substrate oligonucleotides (6-7, 9-12), 100 mM MOPS-buffer at pH 7.2, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO. The reaction plates were heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
After incubation the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added lysate. The plates were then centrifuged at 4,000 rpm for 5 min. Subsequently a 50 pL aliquot of the supernatant was removed from each well and added to a deep well 96-well plate containing 950 pL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 20 pL of the diluted sample into a deep well 96-well plate containing 180 pL of 5 mM EDTA solution (pH 7.0). The samples were diluted a third time by transferring 30 pL of the diluted sample into a deep well 96-well plate containing 150 pL of 5 mM EDTA solution (pH 7.0). The samples were analyzed via RF-MS to determine the activity of the enzyme variants using the analytical method described in Table 6-3. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 288 following pre-incubation at 4 °C are shown in Table 12-1. Selected ligase variants showing a faster product formation of oligonucleotides (2) and (3) relative to SEQ ID NO: 288 following pre-incubation at 30 °C are shown in Table 12-2.
Figure imgf000110_0001
Figure imgf000111_0001
The engineered dsRNA ligase polypeptides represented by the even numbered sequence identifiers of SEQ ID NOs: 602 to 634 comprise an even numbered sequence identifier of SEQ ID NOs: 636 to 668, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 602 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 636. EXAMPLE 13
Comparison of the catalytic activity of the wildtype polypeptide SEQ ID NO: 2 and the engineered polypeptides SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292
The polynucleotides SEQ ID NO: 1 encoding for the wild-type dsRNA ligase from Bacteriophage RB69, Uniprot ID: Q7Y4V8 with the SEQ ID NO: 2 and the engineered polynucleotides SEQ ID NO: 287, SEQ ID NO: 289 and SEQ ID NO: 291 encoding for the most improved variants from example 11 with polypeptide sequences SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292, have been used for SFP production as described in example 5.
The catalytic activity to convert the substrates oligonucleotides (6-7, 9-12) to the desired siRNA product (1) was evaluated under two reaction conditions: Condition 1 (50 mM Tris-buffer at pH 7.5, 1 mM ATP, 5 mM MgCh and 5 mM DTT, containing either 0 g/L, 0.0020 g/L, 0.0039 g/L, 0.0078 g/L, 0.0156 g/L, 0.0313 g/L, 0.0625 g/L, 0.125 g/L, 0.25 g/L, 0.5 g/L, 1 g/L, or 2 g/L of SFP), and Condition 2 (50 mM Tris-buffer at pH 7.0, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO containing either 0 g/L, 0.0049 g/L, 0.0098 g/L, 0.0195 g/L, 0.0391 g/L, 0.0781 g/L, 0.1563 g/L, 0.3125 g/L, 0.625 g/L, 1.25 g/L, 2.5 g/L, or 5 g/L of SFP). The enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well; condition 1 in reaction plate 1 and condition 2 in reaction plate 2. Both reaction plates were heat-sealed and incubated in a thermocycler at 30 °C. Reaction plate 1 was incubated for 2 h and reaction plate 2 was incubated for 24 h.
Following incubation, the plates were subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added SFP. The plates were then centrifuged at 4,000 rpm for 5 min. A 50 pL aliquot of the supernatant from each well of each plate was removed and subsequently diluted in 50 mM EDTA solution (pH 7.0). Reaction plate 1 was diluted 40 x and reaction plate 2 was diluted 2 400 x. The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2.
Comparative data in Figures 2A and 2B show the relative peak area % of siRNA (1) present in the reaction samples assayed under conditions 1 and 2 respectively. Under both conditions 1 and 2, the polypeptides SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292 exhibit improved dsRNA ligase activity over the wild-type polypeptide SEQ ID NO: 2. EXAMPLE 14
Comparison of the catalytic activity of the wildtype polypeptide SEQ ID NO: 2 and the engineered polypeptides SEQ ID NOs: 288 and 632
The polynucleotides SEQ ID NO: 1 encoding for the wild-type dsRNA ligase from Bacteriophage RB69, Uniprot ID: Q7Y4V8 with the SEQ ID NO: 2 and the engineered polynucleotide SEQ ID NO: 287 and SEQ ID NO: 631 encoding for the most improved variant from example 11 and example 12 with polypeptide sequences SEQ ID NO: 288 and SEQ ID NO: 632 respectively, have been used for SFP production as described in example 5.
The catalytic activity to convert the substrate oligonucleotides (6-7, 9-12) to the desired siRNA product (1) and the thermostability of the two enzymes was evaluated by incubating a stock solution of the SFP for 4 h at either 4 or 37 °C prior to setting up the following ligation reaction: 6 mM (each) substrate oligonucleotides (6-7, 9-12), 100 mM MOPS-buffer at pH 7.2, 30 mM ATP, 60 mM MgCh and 10 % (v/v) DMSO. In addition, the ligation reactions contained, either 0 g/L, 0.156 g/L, 0.313 g/L, 0.625 g/L, 1.25 g/L, 2.5 g/L, 5 g/L, or 10 g/L of SFP. The enzyme assays were carried out in 96-well PCR plates, in 100 pL total reaction volume per well. The reaction plate was heat-sealed and incubated in a thermocycler at 30 °C for 24 h.
Following incubation, the plate was subjected to a heat inactivation step (95 °C, 20 min) to quench the reaction and precipitate proteinaceous content of the added SFP. The plate was then centrifuged at 4,000 rpm for 5 min. A 50 pL aliquot of the supernatant from each well of each plate was removed and subsequently diluted 400 x in 50 mM EDTA solution (pH 7.0). The samples were analyzed via HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2.
Comparative data in Figure 3A shows the relative peak area % of siRNA (1) present in the reaction samples. Figure 3B shows the residual enzyme activity following pre -incubation of the SFP at 37 °C for 4 h, expressed relative to the ligation activity of the SFP pre-incubated at 4 °C for 4 h. Under all conditions, the polypeptides SEQ ID NO: 632, exhibits improved dsRNA ligase activity and thermostability over the wild-type polypeptide SEQ ID NO: 2 and engineered polypeptide SEQ ID NO: 288. SUMMARY OF ENGINEERED dsRNA LIGASE POLYPEPTIDE SEQUENCES
Table 13 provides a summary of the nucleic acid and amino acid sequences of the wild-type and engineered dsRNA ligase sequences described herein. The purification tag used in the Examples and reference in table 13 is the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669).
Table 13.
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001

Claims

1. An engineered double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 666, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352,
354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388,
390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424,
426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,
462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496,
498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532,
534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568,
570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638,
640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, and 668; wherein the engineered dsRNA ligase polypeptide:
(a) has dsRNA ligase activity; and
(b) does not the comprise the amino acid sequence of SEQ ID NO: 302.
2. The engineered dsRNA ligase polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 666, 370, 488, 526, 578, 588, 590, and 592.
3. An engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302 under the same ligation reaction conditions, wherein the engineered dsRNA ligase polypeptide does not the comprise the amino acid sequence of SEQ ID NO: 302.
4. The engineered dsRNA ligase polypeptide of claim 3, wherein the ligation reaction conditions include about 1 pM to about 10 mM oligonucleotide fragment, a source of ATP, about 5 mM to about 100 mM divalent cation, and about 0.5 g/L to about 10 g/L engineered dsRNA ligase polypeptide, pH of about 4.0 to about 8.0, and temperature of about 10 °C to about 50 °C. The engineered dsRNA ligase of any preceding claim, wherein:
(a) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218,
X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244,
X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301,
X303, X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID
NO: 302; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A;
X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; XI 14 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A;
X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R; wherein the numbering refers to SEQ ID NO: 302; and/or
(b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; wherein the numbering refers to SEQ ID NO: 302; and/or
(c) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X36, X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; and/or
(d) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X218 and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; and X221 is I; and/or
(e) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X218, X221 and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; and/or
(f) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; and/or
(g) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 E; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; and/or
(h) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X19, X39, X53, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X19 is D; X39 is A; X 53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; and/or
(i) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from: X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X 53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.
6. The engineered dsRNA ligase polypeptide of any of claims 1-5, wherein the engineered dsRNA ligase polypeptide comprises a purification tag; optionally wherein the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 632, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,
196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,
232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,
268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 602,
604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, and 634.
7. A polypeptide immobilized on a solid material by chemical bond or a physical adsorption method, wherein the polypeptide comprises an engineered dsRNA ligase polypeptide according to any one of claims 1-6.
8. A polynucleotide encoding the engineered dsRNA ligase polypeptide of any one of claims 1-6; optionally wherein:
(a) the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs:
303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335,
337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369,
371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403,
405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,
439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471,
473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505,
507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539,
541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573,
575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 635, 637, 639, 641,
643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667; and/or
(b) the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,
199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,
233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,
267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299,
601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, and
633.
9. An expression vector comprising the polynucleotide according to claim 8; optionally wherein the expression vector comprises a plasmid, a cosmid, a bacteriophage or a viral vector.
10. A host cell comprising the polynucleotide of claim 8 or the expression vector of claim 9, optionally wherein the host cell is E. coli.
11. A method of preparing an engineered dsRNA ligase polypeptide, which comprises the steps of culturing the host cell according to claim 10 and obtaining an engineered dsRNA ligase polypeptide from the culture.
12. An engineered dsRNA ligase catalyst obtainable by culturing the host cells according to claim 10, or according to the method of claim 11, wherein said engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptides, or an article processed therewith, wherein the article refers to an extract obtained from the culture of host cell, an isolated product obtained by isolating or purifying an engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing host cell, an extract thereof, or isolated product of the extract.
13. A method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting:
(i) two or more oligonucleotide fragments;
(ii) an engineered dsRNA ligase polypeptide according to any one of claims 1-6;
(iii) a source of ATP; and
(iv) a divalent cation; to obtain an oligonucleotide; optionally wherein:
(a) the method further comprises a step of purifying the oligonucleotide; and/or
(b) the method is performed using a sub-stoichiometric concentration of AMP and/or ATP; and/or
(c) the method is performed with a divalent cation concentration of 5-100 mM, optionally 30-50 mM.
14. Use of the engineered dsRNA ligase polypeptide according to any one of claims 1-6 in the production of an oligonucleotide from two or more oligonucleotide fragments.
15. The method of claim 13 or the use of claim 14, wherein:
(a) the oligonucleotide is up to 60 nucleotides in length; and/or
(b) each of the oligonucleotide fragments are 4-16 nucleotides in length, optionally 6- 9 nucleotides in length; and/or
(c) one or more of the oligonucleotide fragment(s) comprises one or two overhangs; and/or
(d) one or more of the oligonucleotide fragments comprises a chemical modification; optionally wherein the chemical modification is selected from:
(i) a modified backbone, optionally selected from a phosphorothioate (e.g. chiral phosphorothioate) or methylphosphonate intemucleotide linkage;
(ii) a modified nucleotide, optionally selected from 2'-O-methyl (2’-OMe), 2'- flouro (2’-F), 2'-deoxy, 2'-deoxy-2’-fluoro, 2'-O-methoxyethyl (2'-0-M0E), 2'- O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O- DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g. mesyl phosphoramidate), 2',3'-seco nucleotide mimic, 2'-F-arabino nucleotide, abasic nucleotide, 2'- amino modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, vinylphosphonate (e.g. 5’ vinylphosphonate), and cyclopropyl phosphonate deoxyribonucleotide; and/or
(iii) conjugation to a ligand, optionally wherein the ligand comprises one or more N-Acetylgalactosamine (GalNAc) derivatives.
16. A composition comprising: i. the engineered dsRNA ligase polypeptide according to any one of claims 1-6; ii. a source of ATP; and iii. a divalent cation; optionally wherein the composition further comprises two or more oligonucleotide fragments.
17. A kit comprising: i. the engineered dsRNA ligase polypeptide according to any one of claims 1-6; ii. a source of ATP; iii. a divalent cation; and iv. instructions for use in a method of producing an oligonucleotide from two or more oligonucleotide fragments.
18. The method of claim 13 or claim 15, the composition of claim 16 or the kit of claim
17, wherein:
(A) the source of ATP comprises ATP; and/or
(B) the source of ATP comprises:
(a) polyphosphate kinase (PPK);
(b) polyphosphate; and
(c) AMP and/or ATP; optionally wherein:
(i) the PPK is selected from PPK12 or ajPAP; and/or
(ii) the polyphosphate is a polyphosphate salt, optionally wherein the polyphosphate salt is sodium polyphosphate (Maddrell’s salt) or sodium hexametaphosphate (Graham’s salt); and/or
(C) the divalent cation cofactor is Mg2+ or Mn2+.
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