AU7406700A - Design of high affinity rnase h recruiting oligonucleotide - Google Patents
Design of high affinity rnase h recruiting oligonucleotide Download PDFInfo
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Description
WO 01/25248 PCT/DKOO/00550 1 Design of high affinity RNase H recruiting oligonucleotide Field of Invention The present invention relates to the field of bicyclic DNA analogues which are useful for designing oligomers that forms high affinity duplexes with complementary RNA wherein 5 said duplexes are substrates for RNase H. The oligonucleotides may be partially or fully composed of bicyclic DNA analogues. Background of the invention The term "antisense" relates to the use of oligonucleotides as therapeutic agents. Briefly, 10 an antisense drug operates by binding to the mRNA thereby blocking or modulating its translation into protein. Thus, antisense drugs may be used to directly block the synthesis of disease causing proteins. It may, of course, equally well be used to block synthesis of normal proteins in cases where these participate in, and aggravate a pathophysiological process. Also, it ought to be emphasised that antisense drugs can be used to activate 15 genes rather that suppressing them. As an example, this can be achieved by blocking the synthesis of a natural suppressor protein. Mechanistically, the hybridising oligonucleotide is thought to elicit its effect by either cre ating a physical block to the translation process or by recruiting a cellular enzyme (RNase 20 H) that specifically degrades the mRNA part of the mRNA/antisense oligonucleotide du plex. Not unexpectedly, oligonucleotides must satisfy a large number of different requirements to be useful as antisense drugs. Importantly, the antisense oligonucleotide must bind with 25 high affinity and specificity to its target mRNA, must have the ability to recruit RNase H, must be able to reach its site of action within the cell, must be stable to extra - and intra cellular nucleases both endo- and exo-nucleases, must be non-toxic/minimally immune stimulatory, etc. 30 Natural DNA only exhibit modest affinity for RNA and fall short on a number of the other critical characteristics, especially nuclease resistance. Hence, a significant effort has been invested to identify novel analogues with improved antisense properties. In particular the search has focused on identifying novel analogues, which combine an increased affinity WO 01/25248 PCT/DKOO/00550 2 for complementary nucleic acids with the RNase H recruiting ability of natural DNA. Both of these properties have been demonstrated to correlate in a strongly positive manner with biological activity. Of the vast number of analogues that have emerged from this work, only few retain the ability to recruit RNase H and very few provide useful increases 5 in affinity. Sadly, those that do provide a useful increase in affinity fail to recruit RNase H. In the face of these results the field have turned to mixed backbone oligonucleotides as a means to provide higher potency antisense drugs, i.e. antisense molecules that operates by a two fold mechanism of action 1) high affinity mediated translational arrest at the ribo 10 somal level and 2) activation of RNase H. These molecules (termed gab-mers) typically comprise a central region of at least six contiguous, low affinity phosphorothioates (RNase H recruiting analogues) flanked by stretches of high affinity analogues (non RNase H re cruiting analogues) that enhance the ability to promote translational arrest. Although ex pected to out-perform current phosphorothioate antisense drugs, the gab-mers are not 15 considered the ideal antisense molecules. Amongst their weaknesses is the requirement for a rather fixed design and the presence of high and low affinity domains within the molecule, which may compromise biological activity. The enzyme RNase H selectively binds to heterogeneous DNA/RNA duplexes and de 20 grades the RNA part of the duplex. Homogeneous DNA/DNA and RNA/RNA duplexes, which only differs molecularly from the DNA/RNA duplex at the 2' position (DNA/DNA: 2' H/2'-H; RNA/RNA: 2'-OH/2'-OH and DNA/RNA: 2'-H/2'-OH) are not substrates for the enzyme. This suggests that either the molecular composition at the 2' position itself or the structural feature it imposes on the helix is vital for enzyme recognition. Consistent with 25 this notion, all 2'-modified analogues that have so far been reported to exhibit increased affinity have lost the ability to recruit RNase H. Detailed description of the invention. Locked Nucleic Acid (LNA) is a novel, nucleic bicyclic acid analogue in which the 2'- and 30 4' position of the furanose ring are linked by an O-methylene (oxy-LNA), S-methylene (thio-LNA) or NH 2 -methylene moiety (amino-LNA). This linkage restricts the conforma tional freedom of the furanose ring and leads to an increase in affinity which is by far the highest ever reported for a DNA analogue (WO 99/14226). 35 WO 01/25248 PCT/DKOO/00550 3 Despite the fact that the modification in LNA involves the 2'-position we have found that the activity of RNase H is not dependent on a contiguous stretch of DNA or phosphoro thioated bases when oxy-LNA is used as a component of the oligonucleotide. In fact, we have found that oligonucleotides composed entirely of oxy-LNA are able to recruit RNase 5 H. Oxy-LNA oligonucleotides thus constitutes the first ever DNA analogue to display the long sought after combination of very high affinity and ability to recruit RNase H. The im plications are that oxy-LNA by itself may be used to construct novel antisense molecules with enhanced biological activity. Alternatively, oxy-LNA may be used in combination with non-oxy-LNA, such as standard DNA, RNA or other analogues, e.g. thio-LNA or amino 10 LNA to create high affinity, RNase H recruiting antisense compounds without the need to adhere to any fixed design. An "oxy-LNA monomer" is defined herein as a nucleotide monomer of the formula la
R
5
R
5 * P-~ B R 4*''' '"R1* la R3 R2 15
R
3 * R2 wherein X is oxygen; B is a nucleobase; R 1 *, R 2 , R 3 , R 5 and R 5 * are hydrogen; P desig nates the radical position for an internucleoside linkage to a succeeding monomer, or a 5' terminal group, R3* is an internucleoside linkage to a preceding monomer, or a 3'-terminal 20 group; and R 2 and R 4 * together designate -O-CH 2 - where the oxygen is attached in the 2' position. The term "nucleobase" covers the naturally occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally occuring nucleo 25 bases such as xanthine, diaminopurine, 8-oxo-N -methyladenine, 7-deazaxanthine, 7 deazaguanine, N 4 , N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C 3
-C
6 )-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5 methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S. Pat No. 5,432,272 and Susan M. Freier and 30 Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol. 25, pp 4429-4443. The term "nu cleobase" thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. It should be clear to the person skilled in WO 01/25248 PCT/DKOO/00550 4 the art that various nucleobases which previously have been considered "non-naturally occurring" have subsequently been found in nature. A "non-oxy-LNA" monomer is broadly defined as any nucleoside (i.e. a glycoside of a het 5 erocyclic base) which is not itself an oxy-LNA but which can be used in combination with oxy-LNA monomers to construct oligos which have the ability to bind sequence specifi cally to complementary nucleic acids. Examples of non-oxy-LNA monomers include 2' deoxynucleotides (DNA) or nucleotides (RNA) or any analogues of these monomers which are not oxy-LNA, such as for example the thio-LNA and amino-LNA described by 10 Wengel and coworkers (Singh et al. J. Org. Chem. 1998, 6, 6078-9, and the derivatives described in Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443. It should be understood that the incorporation of non-oxy-LNA monomer(s) into an oxy 15 LNA oligo may change the RNAseH recruiting characteristics of the oxy-LNA/non-oxy LNA chimeric oligo. Thus, depending on the number and type of non-oxy-LNA mono mer(s) used, and the position of these monomers in the resulting oxy-LNA/non-oxy-LNA chimeric oligo, the chimera may have an increased, unaltered or decreased ability to re cruit RNAsdeH as compared to the corresponding all oxy-LNA oligo. 20 As mentioned above, a wide variety of modifications of the deoxynucleotide skeleton can be contemplated and one large group of possible non-oxy-LNA can be described by the following formula I
R
5
R
5 * B R4- R1 | R R2 25
R
3 * R 2 * wherein X is -0-; B is selected from nucleobases; R 1 is hydrogen; P designates the radical position for an internucleoside linkage to a succeeding monomer, 30 or a 5'-terminal group, such internucleoside linkage or 5'-terminal group optionally includ ing the substituent R 5 , R 5 being hydrogen or included in an internucleoside linkage, WO 01/25248 PCT/DKOO/00550 5
R
3 * is a group P* which designates an internucleoside linkage to a preceding monomer, or a 3'-terminal group; one or two pairs of non-geminal substituents selected from the present substituents of R 2 , 5 R 2 , R 3 , R 4 *, may designate a biradical consisting of 1-4 groups/atoms selected from -C(RaRb)-, -C(Ra)=C(Ra)-, -C(Ra)=N-, -O-, -S-, -SO 2 -, -N(Ra)-, and >C=Z, wherein Z is selected from -0-, -S-, and -N(Ra)-, and Ra and Rb each is independ ently selected from hydrogen, optionally substituted C 1
.
6 -alkyl, optionally substi tuted C 2
-
6 -alkenyl, hydroxy, C 1 6 -alkoxy, C 2
-
6 -alkenyloxy, carboxy, C1.
6 -alkoxy 10 carbonyl, C 16 -alkylcarbonyl, formyl, amino, mono- and di(C 1
.
6 -alkyl)amino, car bamoyl, mono- and di(C 1
.
6 -alkyl)-amino-carbonyl, amino-C 1
.
6 -alkyl-aminocarbonyl, mono- and di(C 1
.
6 -alkyl)amino-C 1
.
6 -alkyl-aminocarbonyl, C 1
.
6 -alkyl-carbonylamino, carbamido, C 1
.
6 -alkanoyloxy, sulphono, C 1
.
6 -alkylsulphonyloxy, nitro, azido, sul phanyl, C 1
.
6 -alkylthio, halogen, photochemically active groups, thermochemically 15 active groups, chelating groups, reporter groups, and ligands, said possible pair of non-geminal substituents thereby forming a monocyclic entity to gether with (i) the atoms to which said non-geminal substituents are bound and (ii) any intervening atoms; and 20 each of the substituents R 2 , R 2 , R 3 , R4* which are present and not involved in the possible biradical is independently selected from hydrogen, optionally substituted C 1
.
6 -alkyl, op tionally substituted C2- 6 -alkenyl, hydroxy, C 1
.
6 -alkoxy, C2- 6 -alkenyloxy, carboxy, C1.6 alkoxycarbonyl, C 1
.
6 -alkylcarbonyl, formyl, amino, mono- and di(C 1
.
6 -alkyl)amino, car bamoyl, mono- and di(C 1
.
6 -alkyl)-amino-carbonyl, amino-C 1
.
6 -alkyl-aminocarbonyl, mono 25 and di(C 1
.
6 -alkyl)amino-C 1 e-alkyl-aminocarbonyl, C 1
.
6 -alkyl-carbonylamino, carbamido,
C
1
.
6 -alkanoyloxy, sulphono, C 1
.
6 -alkylsulphonyloxy, nitro, azido, sulphanyl, C 1
.
6 -alkylthio, halogen, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands; 30 and basic salts and acid addition salts thereof; with the proviso the monomer is not oxy-LNA. Particularly preferred non-oxy-LNA monomers are 2'-deoxyribonucleotides, ribonucleo 35 tides, and analogues thereof that are modified at the 2'-position in the ribose, such as 2'- WO 01/25248 PCT/DKOO/00550 6 O-methyl, 2'-fluoro, 2'-trifluoromethyl, 2'-O-(2-methoxyethyl), 2'-O-aminopropyl, 2'-0 dimethylamino-oxyethyl, 2'-O-fluoroethyl or 2'-O-propenyl, and analogues wherein the modification involves both the 2'and 3' position, preferably such analogues wherein the modifications links the 2'- and 3'-position in the ribose, such as those described by Wen 5 gel and coworkers (Nielsen et al., J. Chem. Soc., Perkin Trans. 1, 1997, 3423-33, and in WO 99/14226), and analogues wherein the modification involves both the 2'and 4' posi tion, preferably such analogues wherein the modifications links the 2'- and 4'-position in the ribose, such as analogues having a -CH 2 -S- or a -CH 2 -NH- or a -CH 2 -NMe- bridge (see Wengel and coworkers in Singh et al. J. Org. Chem. 1998, 6, 6078-9). Although, 10 non-oxy-LNA monomers having the p-D-ribo configuration are often the most applicable, further interesting examples (and in fact also applicable) of non-oxy-LNA are the stereoi someric of the natural B-D-ribo configuation. Particularly interesting are the ax-L-ribo, the p D-xylo and the a-L-xylo configurations (see Beier et al., Science, 1999, 283, 699 and Es chenmoser, Science, 1999, 284, 2118), in particular those having a 2'-4' -CH 2 -S-, -CH 2 15 NH-, -CH 2 -0- or -CH 2 -NMe- bridge (see Wengel and coworkers in Rajwanshi et al., Chem. Commun., 1999, 1395 and Rajwanshi et al., Chem. Commun., 1999, submitted) In the present context, the term "oligonucleotide" which is the same as "oligomer" which is the same as "oligo" means a successive chain of nucleoside monomers (i.e. glycosides of 20 heterocyclic bases) connected via internucleoside linkages. The linkage between two successive monomers in the oligo consist of 2 to 4, preferably 3, groups/atoms selected from -CH 2 -, -0-, -S-, -NRH-, >C=O, >C=NRH, >C=S, -Si(R") 2 -, -so-, -S(0) 2 -, -P(O) 2 -,
-PO(BH
3 )-, -P(O,S)-, -P(S) 2 -, -PO(R")-, -PO(OCH 3 )-, and -PO(NHRH)-, where RH is se lected form hydrogen and ClA-alkyl, and R" is selected from C 1
.
6 -alkyl and phenyl. Illu 25 strative examples of such linkages are -CH 2
-CH
2
-CH
2 - -CH 2
-CO-CH
2 - -CH 2
-CHOH-CH
2 -O-CH 2 -O-, -0-CH2-CH 2 -, -O-CH 2 -CH= (including R 5 when used as a linkage to a suc ceeding monomer), -CH 2
-CH
2 -O-, -NRH-CH 2
-CH
2 -, -CH 2
-CH
2 -NRH-, -CH 2 -N RH-CH 2 -,
-O-CH
2
-CH
2 -NRH-, -NRH-CO-O-, -NRHCO-NRH-, -NRH-CS-NRH-, -NRH-C(=NRH)-NRH_ -NRH-CO-CH2-NRH-, -0-CO-O-, -O-CO-CH 2 -0-, -O-CH 2 -CO-O-, -CH 2 -CO-NRH_, 000 30 NRH-, -NRH-CO-CH 2 -, -O-CH2-CO-NRH-, -O-CH 2
-CH
2 -NRH-, -CH=N-O-, -CH 2 -NRH-O-,
-CH
2 -0-N= (including R 5 when used as a linkage to a succeeding monomer), -CH 2 -0 NRH-, -CO-NRH-CH 2 -, -CH 2 -NRH-O-, -CH 2 -N RH-CO-, -O-NRH-CH 2 -, -O-NRH-, -O-CH 2 -S-,
-S-CH
2 -0-, -CH 2
-CH
2 -S-, -0-CH 2
-CH
2 -S-, -S-CH 2 -CH= (including R 5 when used as a link age to a succeeding monomer), -S-CH 2
-CH
2 -, -S-CH 2
-CH
2 -0-, -S-CH 2
-CH
2 -S-, -CH 2
-S
35 CH 2 -, -CH 2
-SO-CH
2 -, -CH 2
-SO
2
-CH
2 -, -0-SO-O-, -0-S(0) 2 -0-, -O-S(O) 2
-CH
2 -, -0-S(0) 2
-
WO 01/25248 PCT/DKOO/00550 7 N RH-, -N RH-S(0) 2
-CH
2 -, -O-S(O) 2
-CH
2 -, -O-P(O) 2 -O-, -O-P(O,S)-O-, -O-P(S) 2 -O-,
-S-P(O)
2 -O-, -S-P(O,S)-O-, -S-P(S) 2 -0-, -O-P(O) 2 -S-, -O-P(0,S)-S-, -O-P(S) 2 -S-,
-S-P(O)
2 -S-, -S-P(O,S)-S-, -S-P(S) 2 -S-, -O-PO(R")-O-, -O-PO(OCH 3 )-O-, -O-PO
(OCH
2
CH
3 )-O-, -O-PO(OCH 2
CH
2 S-R)-O-, -O-PO(BH 3 )-O-, -O-PO(NHRN)_O_, -O-p(O) 2 5 NRH-, -NRHP()2-0-, -O-P(O,NRH)-O-, -CH 2
-P(O)
2 -O-, -O-P(O) 2
-CH
2 -, and -O-Si(R") 2 -O-; among which -CH 2 -CO-NRH-, -CH 2 -NRH-O, -S-CH 2 -O-, -O-P(O) 2 -O-, -O-P(O,S)-O-,
-O-P(S)
2 -O-, -NRH~P(O)2-0-, -O-P(O,NRH)-O-, -O-PO(R")-O-, -O-PO(CH 3 )-O-, and -O-PO(NHRN)-O-, where RH is selected form hydrogen and ClA-alkyl, and R" is selected from C 1
.
6 -alkyl and phenyl, are especially preferred. Further illustrative examples are 10 given in Mesmaeker et. al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429 4443. The left-hand side of the internucleoside linkage is bound to the 5-membered ring as substituent P at the 3'-position, whereas the right-hand side is bound to the 5'-position of a preceding monomer. 15 The term "succeeding monomer" relates to the neighbouring monomer in the 5'-terminal direction and the "preceding monomer" relates to the neighbouring monomer in the 3' terminal direction. 20 Monomers are referred to as being "complementary" if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with C, A with T or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, pseudoisocytosine with G, etc. 25 When the modified oxy-LNA oligo contain at least two non-oxy-LNA monomers these may contain the same or different nucleobases at the 1'-position and be identical at all other positions or they may contain the same or different nucleobases at the 1'-position and be non-identical at at least one other position. 30 Accordingly, the present invention describes a method for degrading RNA in-vivo (in a cell or organism) or in-vitro by providing a high affinity oligonucleotide which activates RNaseH when the high affinity oligonucleotide is hybridised to a complementary RNA tar get sequence, said high affinity oligonucleotide may consist of oxy-LNA monomers exclu sively. 35 WO 01/25248 PCT/DKOO/00550 8 Alternatively, the high affinity oligonucleotide may also consist of both oxy-LNA and non oxy-LNA monomers, in this case the high affinity oligonucleotide contains at the most five, e.g. 4, e.g. 3 , e.g. 2 contiguous non-oxy-LNA monomers at any given position in the oli gonucleotide, e.g. said high affinity oligonucleotide consists of both oxy-LNA and non-oxy 5 LNA monomers, wherein none of the non-oxy-LNA monomers are located adjacent to each other. The high affinity oligonucleotide may also contain one or more segments of contigous non-oxy-LNA monomers. For instance, a stretch of contigous non-oxy-LNA monomers 10 may be located in the centre of the oligonucleotide and with flanking segments consisting of oxy-LNA monomers. Alternatively the stretch of contigous non-oxy-LNA monomers may be located at either or both ends. Also, the oxy-LNA segement(s) may be either contigous or interrupted by 1 or more non-oxy-LNA monomers. Also, the high affinity oligonucleotide may comprise more than one type of internucleoside linkage such as for example mixes 15 of phosphordiester and phosphorothioate linkages. The resulting high affinity oligo containing oxy-LNA monomers and/or non-oxy-LNA mono mers can thus be characterized by the general formula 20 5'-[XmYnXplq-3' X is oxy-LNA and Y is non-oxy-LNA, wherein m and p are integers from 0 to 30, n is an integer from 0 to 3 and q is an integer from 1 to 10 with the proviso that the sum of m+n+p multiplied with q is in the range of 6-100, such as 8, e.g. 9, e.g. 10, e.g. 11, e.g. 12, e.g. 25 13, e.g. 14, e.g. 15, e.g. 16, e.g. 17, e.g. 18, e.g. 19, e.g. 20, e.g. 21, e.g. 22, e.g. 23, e.g. 24, e.g. 25, e.g. 26, e.g. 27, e.g. 28, e.g. 29, e.g. 30, e.g. 35, e.g. 40, e.g. 45, e.g. 50, e.g. 60, e.g. 70, e.g. 80, e.g. 90, such as 100. The present invention provides oligos which combine high affinity and specificity for their 30 target RNA molecules with the ability to recruit RNAseH to an extend that makes them useful as antisense therapeutic agents. The oligos may be composed entirely of oxy-LNA monomers or they may be composed of both oxy and non-oxy-LNA monomers. When both oxy-LNA and non-oxy-LNA monomer(s) are present in the oligo, the RNAseH 35 recruiting characteristics of the chimeric oligo may be similar to, or different from, that of WO 01/25248 PCT/DKOO/00550 9 the corresponding oxy-LNA oligo. Thus, in one aspect of the invention, non-oxy-LNA monomer(s) is/are used in such a way that they do not change the RNAseH recruting characteristics of the oxy-LNA/non-oxy-LNA chimeric oligo compared to the correspond ing all oxy-LNA oligo. In another aspect of the invention the non-oxy-LNA monomer(s) 5 is/are used purposely to change the RNAseH recruiting characteristics of an oxy-LNA oligo, either increasing or decreasing its efficiency to promote RNAseH cleavage when hybridised to its complementary RNA target compared to the corresponding all oxy-LNA oligo. 10 When both oxy-LNA and non-oxy-LNA monomer(s) is/are present in the oligo, the ability of the chimeric oligo to discriminate between its complementary target RNA and target RNAs containing one or more Watson-Crick mismatches may be different from the ability of the corresponding all oxy-LNA oligo to discriminate between its matched and mis matched target RNAs. For instance, the ability of an oxy-LNA oligo to discriminate be 15 tween a complementary target RNA and a single base mismatched target RNA can be enhanced by incorporating non-oxy-LNA monomer(s), such as for instance DNA, RNA, thio-LNA or amino-LNA, either at, or close to, the mismatched position as described in applicant's Danish patent application entitled "Metod of increasing the specificity of oxy LNA oligonuclotides" filed on the same day as the present application. Thus, in another 20 aspect of the invention non-oxy-LNA monomer(s) is/are used purposely to construct an oxy-LNA/non-oxy-LNA oligo which exhibit increased specificity but unaltered RNAseH re cruiting characteristics compared to the corresponding all oxy-LNA oligo. In another as pect of the invention the non-oxy-LNA monomer(s) is/are used purposely to construct an oxy-LNA/non-oxy-LNA oligo which exhibit both increased specificity and altered RNAseH 25 recruiting characteristics compared to the corresponding all oxy-LNA oligo Additionally, the oligonucleotide of the present invention may be conjugated with com pounds selected from proteins, amplicons, enzymes, polysaccharides, antibodies, hap tens and peptides. 30 WO 01/25248 PCT/DKOO/00550 10 Examples Example 1: LNA containing oligonucleotides recruit RNase H Two 41-mer oligonucleotides, that make up a linearised double-stranded template for subsequent T7 polymerase run-off transcription, were used to obtain target RNA corre 5 sponding to the following 15mer oligonucleotides: DNA control; 5'- gtgtccgagacgttg-3' phosphorothioate control; 5'-gtgtccgagacgttg-3' LNA qab-mer; 5'-GTGTccgagaCGTTG-3' (LNA in capital letters, DNA is small letters) and 10 LNA-mix-mer; 5'-gTgTCCgAgACgTTg-3' (LNA in capital letters, DNA is small letters) In the 5'end of the sense template strand, the promoter sequence for T7 polymerase rec ognition and initiation of transcription were contained, followed by the DNA sequence coding for the target-RNA sequence. The two complementary oligonucleotides were 15 heated to 800C for 10min to produce the linearised double-strand template. A 2 0pl in vitro transcription reaction containing 500iM each of ATP, GTP and CTP, 12pM of UTP, ap prox. 50ptCi of a- 32 p UTP, 1 x transcription buffer (Tris-HCI, pH 7.5), 10mM dithiotretiol, 1% BSA, 20 U of RNasin ribonuclease inhibitor, 0.2 l template and 250 units T7 RNA polymerase. The inclusion of RNasin inhibitor was to prevent degradation of the target 20 RNA from ribonucleases. The reactions were carried out at 370C for 2h to produce the desired 24mer 32 U-labelled RNA run-off transcript. For target RNA purification, 1.5il (1.5 Units) of DNase I was added to the RNA which was resolved in a 15% polyacrylamide gel containing 7M urea and the correctly sized fragment was excised from the gel, dispensed in elution buffer (0.1% SDS, 0.5M ammonium acetate, 10mM Mg-acatate) and incubated 25 at room temperature overnight. The target RNA sequence was then purified via ethanol precipitation, the supernatants filtered through a Millipore (0.45m) and collected by etha nol precipitation. The pellets were diluted in TE-buffer and subsequently subjected to RNAse H digestion assay. Herein, the decrease of intact substrate, i.e. the 24-mer a- 32 p UTP labelled target RNA sequence, was assayed over time as follows. The reactions 30 were carried out in a total volume of 1 10ptl and contained (added in the order mentioned): 1 x nuclease-free buffer (20mM Tris-HCI, pH 7.5, 40mM KCI, 8mM MgCl 2 , 0.03 mg/ml BSA), 10mM dithiotretiol, 4% glycerol, 1OOnM of oligonucleotide, 3 Units RNasin inhibitor, labelled target RNA strand and 0.1 U of RNase H. An excess of oligonucleotide was added to each reaction to ensure full hybridisation of the RNA target sequences. Two WO 01/25248 PCT/DKOO/00550 11 negative controls were also included and were prepared as above but (1) without any oli gonucleotide, or (2) without RNase H added to the reaction mixture. All the reactions were incubated at 37 0 C. At time points 0, 10, 20, 40 and 60 min., 10lt aliquots were taken and immediately added to ice-cold formamide loading buffer to quench the reaction and stored 5 at -20 0 C. The samples were heated to 85 0 C for 5 min. prior to loading and running on a 15% polyacrylamide gel containing 7M urea. The gels were vacuum dried and exposed to autoradiographic films over night and subsequently subjected to densitometric calcula tions using the Easy Win imaging software (Hero Labs). The volume density of intact tar get RNA were calculated in each lane with correction for background. The volume density 10 for the time zero sample was set as reference value for each incubation. Relative values for the other time-points samples in the corresponding incubation were calculated based on these reference values. Brief description of figures 15 Figure 1 shows the results of the RNase H experiments. As expected the control DNA and phosphorothioate oligonucleotides both recruit RNAse H very efficiently. Also, as expect ed the LNA oligonucleotide which contains a contiguous stretch of six DNA monomers in the middle (LNA gab-mer) recruits RNAse H efficiently. Surprisingly, however, the LNA mix-mer which contains only single DNA monomers interdispersed between LNA mono 20 mers also recruits RNAse H. We conclude that the activity of RNase H is not contingent on a contiguous stretch of DNA or phosphorothioated bases when LNA is used as a com ponent of the oligonucleotide.
Claims (23)
1. A method for degrading RNA comprising providing a high affinity oligonucleotide which recruits RNaseH when hybridised to an RNA target sequence, wherein said high affinity oligonucleotide consists of oxy-LNA monomers exclusively. 5
2. A method for degrading RNA comprising providing a high affinity oligonucleotide which recruits RNaseH when hybridised to an RNA target sequence, wherein said high affinity oligonucleotide consists of both oxy-LNA and non-oxy-LNA monomers and wherein there are at the most five contiguous non-oxy-LNA monomers at any given position in the oligo 10 nucleotide.
3. A method for degrading RNA comprising providing a high affinity oligonucleotide which recruits RNaseH when hybridised to an RNA target sequence, wherein said high affinity oligonucleotide consists of both oxy-LNA and non-oxy-LNA monomers and wherein none 15 of the non-oxy-LNA monomers are located adjacent to each other.
4. A method according to any of claims 2 or 3, wherein the presence of the non-oxy-LNA monomer(s) in the oxy-LNA/non-oxy-LNA oligo does not change the RNAseH recruiting characteristics of the oligo compared to the corresponding oxy-LNA oligo. 20
5. A method according to claim 4, wherein the presence of the non-oxy-LNA monomer(s) in the oxy-LNA/non-oxy-LNA oligo modifies the RNAseH recruiting characteristics of the oligo compared to the corresponding oxy-LNA oligo. 25
6. A method according to claim 5, wherein the presence of the non-oxy-LNA monomer(s) in the oxy-LNA/non-oxy-LNA oligo either enhances or reduces the ability of the oligo to recruit RNAseH compared to the corresponding oxy-LNA oligo.
7. A method according to any of the claims 2 to 6 wherein the presence of the non-oxy 30 LNA monomer(s) in the oxy-LNA/non-oxy-LNA oligo increases the ability of the oligo to discriminate between its complementary target RNA and target RNAs containing one or more Watson-Crick mismatches compared to the corresponding oxy-LNA oligo. WO 01/25248 PCT/DKOO/00550 13
8. A method according to any of the previous claims wherein the oligonucleotide is char acterised by the general formula 5'-[XmYnXp]q-3' 5 wherein X is oxy-LNA and Y is non-oxy-LNA, wherein m and p are integers from 0 to 30, n is an integer from 0 to 5 and q is an integer from 1 to 10.
9. A method according to claim 2-8, wherein the non-oxy-LNA monomer(s) is/are deoxyri 10 bonucleotide(s).
10. A method according to claim 9, wherein the deoxyribonucleotide is modified at the 2' position in the ribose. 15
11. A method according to claim 10, wherein the 2'-modification is a hydroxyl, 2'-0 methyl, 2'-fluoro, 2'-trifluoromethyl, 2'-O-(2-methoxyethyl), 2'-O-aminopropyl, 2'-O dimethylamino-oxyethyl, 2'-O-fluoroethyl or 2'-O-propenyl.
12. A method according to claim 11, wherein the modification also involves the 3' position, 20 preferably modifications that links the 2'- and 3'-position in the ribose.
13. A method according to claim 12, wherein the modification also involves the 4' position, preferably modifications that links the 2'- and 4'-position in the ribose. 25
14. A method according to claim 13, wherein the modification is selected from the group consisting of a 2'-4' link being a -CH 2 -S-, -CH 2 -NH-, or -CH 2 -NMe- bridge.
15. A method according to any of the claims 9 to 14, wherein the nucleotide has the C-D ribo, p-D-xylo, or a-L-xylo configuration. 30
16. A method according to any of the claims 9 to 15, wherein either all or some of the oxy LNA monomers or all or some of the non-oxy-LNA monomer(s) or all or some of both the oxy-LNA monomers and non-oxy-LNA monomer(s) contain a 3'- or 5'- modification that results in an internucleoside linkage other than the natural phosphorodiester linkage. 35 WO 01/25248 PCT/DKOO/00550 14
17. A method according to claim 16, wherein the modification is selected from the group consisting of -O-P(O) 2 -O-, -O-P(O,S)-O-, -O-P(S) 2 -O-, -NRH-P(O) 2 -O-, -O-P(O,NR H)-O_, -O-PO(R")-O-, -O-PO(CH 3 )-O-, and -O-PO(NHRN)-O-, where RH is selected form hydro gen and ClA-alkyl, and R" is selected from C 1 . 6 -alkyl and phenyl. 5
18. A method according to any of the preceding claims, wherein the incorporation of the at least one non-oxy-LNA monomer changes the affinity of the resulting oligo towards its complementary nucleic acid compared to the affinity of the all-oxy-LNA oligo by a ATm of no more than ± 5 0 C. 10
19. A method according to claim 18, wherein the affinity is changed by no more than ± 100C.
20. A method according to any of claims 18 or 19, wherein at least two non-oxy-LNA 15 monomers containing either the same or different nucleobases at the 1'-position and be ing identical at all other positions are used.
21. A method according to any of claims 18 or 19, wherein at least two non-oxy-LNA monomers containing either the same or different nucleobases at the 1'-position and be 20 ing non-identical in at least one other position are used.
22. A oligomer according to any of the previous claims, wherein said oligomer is used as a therapeutic compound, e.g. as an antisense compound. 25
23. An oligomer as defined in any of the previous claims, which is conjugated with com pounds selected from proteins, amplicons, enzymes, polysaccharides, antibodies, hap tens, and peptides.
Applications Claiming Priority (5)
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| US15772499P | 1999-10-05 | 1999-10-05 | |
| US60157724 | 1999-10-05 | ||
| PCT/DK2000/000550 WO2001025248A2 (en) | 1999-10-04 | 2000-10-03 | Design of high affinity rnase h recruiting oligonucleotide |
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| AU7406700A true AU7406700A (en) | 2001-05-10 |
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| AU74067/00A Abandoned AU7406700A (en) | 1999-10-04 | 2000-10-03 | Design of high affinity rnase h recruiting oligonucleotide |
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| EP (1) | EP1224280A2 (en) |
| JP (1) | JP2003511016A (en) |
| AU (1) | AU7406700A (en) |
| CA (1) | CA2385853A1 (en) |
| IL (1) | IL148916A0 (en) |
| WO (1) | WO2001025248A2 (en) |
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| US5700922A (en) * | 1991-12-24 | 1997-12-23 | Isis Pharmaceuticals, Inc. | PNA-DNA-PNA chimeric macromolecules |
| KR100414936B1 (en) * | 1997-09-12 | 2004-01-13 | 엑시콘 에이/에스 | Bi- and tri-cyclic nucleoside, nucleotide and oligonucleotide analogues |
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- 2000-10-03 WO PCT/DK2000/000550 patent/WO2001025248A2/en not_active Application Discontinuation
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| EP1224280A2 (en) | 2002-07-24 |
| WO2001025248A2 (en) | 2001-04-12 |
| CA2385853A1 (en) | 2001-04-12 |
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| IL148916A0 (en) | 2002-09-12 |
| WO2001025248A3 (en) | 2001-08-30 |
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