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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/30—Nucleotides
  • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • C—CHEMISTRY; METALLURGY
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1241—Nucleotidyltransferases (2.7.7)
  • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• • C—CHEMISTRY; METALLURGY
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6869—Methods for sequencing

Definitions

• the instant invention pertains to nucleic acid terminators which result in improved sequence data.
• the instant invention also pertains to charge modified nueleie aeid terminators which allow for the direct loading of nucleic acid sequencing reactions onto separating media.
• the sequence of nucleotide bases in a DNA molecule can be determined in a variety of ways.
• the chain termination method generally involves synthesizing DNA complementary to the template strand to be sequenced by extending a primer able to hybridize to a portion of that te ⁇ plate strand with a DNA polymerase. During the synthesis reaction, deoxynucleoside triphosphates (dNTP's) are incorporated to form a DNA fragment until a chain terminating agent, for example, a dideoxynucleotide triphosphate (ddNTP) is incorporated. Incorporation of a ddNTP prevents further DNA synthesis (a process called chain termination).
• each DNA fragment synthesized in this procedure is then determined by gel electrophoresis and this information used to determine the sequence of nucleotides in the original template DNA-
• Tabor and Richardson U.S. Patent No. 4,795,699, the entire disclosure of which is incorporated herein, describe a two step sequencing method in which an unlabeled primer is labeled in a labeling step, and then extended in the presence of excess dNTPs and a ddNTP in a chain termination step.
• a low concentration of dNTPs is provided (one being labeled) to allow a small amount of primer extension.
• the primer may be labeled, for example with P " , by a process using a polynucleotide kinase.
• P polynucleotide kinase
• a labeled -dNTP may be incorporated during the process of DNA synthesis, and the presence of such labeled dNTPs detected by autoradiography or other me ns.
• the dNTP may be labeled either radioactively with P 32 or S 35 .
• 1he primer can be labeled with one or more fluorescent moieties for detection by fluorescence.
• the ddNTP may be labeled, for example, with a fluorescent marker.
• the terminators In a sequencing reaction, the terminators partially decompose, most likely due to the thermocycl ing, conditions, and generate labeled by-products which migrate in the separating media, thus interfering with interpretation of the true sequencing fragments. For example, terminator decomposition products and unreacted te ⁇ mnator inay appear on sequencing gels or electropherograms as peaks or blobs ( _g ⁇ Figure 1, lanes 3 and 4 which show the blobs which result when sequencing reaction products containing conventional terminators are directly loaded onto an electrophoretic gel).
• One aspect of the instant disclosure pertains to a charge ⁇ modif ⁇ ed nucleic acid terminator comprising structure (I)
• Z is mono-, di or triphosphate or thiophosphate, or corresponding boranophosphate X is O, CH 2 , S, or NH;
• S is a sugar or a sugar analogue
• B is a naturally occurring or a synthetic base
• L is alkyl, alkenyL, or alkynyl and is optionally substituted with a reporter moiety
• L, B, S, X, or Z are substituted with a moiety which imparts a net negative charge or a net positive charge to structure (I) at physiological or nucleic acid sequencing conditions.
• the instant disclosure pertains to methods of sequencing nucleic acids using the above charge-modified terminators. In yet another aspect, the instant disclosure pertains to methods of inhibiting a virus which comprises containing a cell infected with a virus with a virus-inhibiting amount of the above charge-modified terminator.
• the instant invention pertains to nucleic acid terminators, which along with the corresponding nucleic acid terminator decomposition products, migrate on separation media at different rates than the sequencing reaction products and which result in improved sequence data.
• nucleic acid terminators also allow for the direct loading of nucleic acid sequencing reactions onto separating media.
• negatively or positively charged moieties are attached to the terminator molecule.
• the unreacted or degraded terminators containing such charged moieties move faster (negatively charged) or in the reverse direction (positively charged) than the DNA sequencing products.
• Z is mono-, di or triphosphate or thiophosphate, or corresponding boranophosphate
• S is a sugar or a sugar analogue
• B is a naturally occurring or a synthetic base
• L is alkyl, alkenyL or alkynyl and is optionally substituted with a reporter moiety; and L, B, S, X, or Z are substituted with a moiety which imparts a net negative charge or a net positive charge to structure (I) at physiological or nucleic acid sequencing conditions.
• the base may be any naturally occurring or synthetic base such as A, T, G, or C or analogs thereof such as 7-deazapurine, inosine, universal bases, etc.
• Suitable base analogs include those disclosed in WO 99/06422 and WO 97/28177.
• the sugar may be furanose, hexose, mono-di-triphosphates, morpholine, didehydro, dideoxyribose, deoxyribose, dioxalone, oxathialane, and analogs thereof.
• the linker may be alkyl, alkenyl, or alkynyl and may contain 1 to about 100 atoms and may contains atoms such as C, H, N, O, S and halogen. In general, the linker contains about 2 to about 50 atoms. Preferably, when the terminator molecule contains a net positive charge, the linker contains about 2 to about 25 atoms, more preferably, the linker contains about 11 to about 25 atoms. Preferably, when the terminator molecule contains a net negative charge, the linker contains about 11 to 25 atoms, more preferably, the linker contains about 18 to about 25 atoms.
• the linker may optionally be substituted with a label, (also referred to as a "reporter or signal moiety.")
• the label may be a moiety such as a radioisotope, electrochemical tag, fluorescent tags, energy transfer (ET) labels, mass spectrometry tags, Raman tags, hapten, chemilluminescent group, enzyme, chromophore, and two or more labels.
• the label may also be charged, e.g. Cy5.5, bis-sulf nated carboxyfluorescein, or a dye attached to a charged moiety, e_g., carboxyfluorescein attached to cysteic acid or similar charged species.
• the terminator molecule is substituted with a moiety which imparts a net negative charge or a net positive charge to structure (I) at physiological or nucleic acid sequencing conditions.
• the moiety may be any charged species which alters the electrophoretic mobility of structure and degradation products, e.g., ⁇ -sulfb- ⁇ -alanine, cysteic acid, sulfonic acids, carboxylates, phosphates, phosphodiesters, phosphonates, amines, quaternised amines, and phosphonium moieties.
• the moiety may be attached to the terminator molecule between the linker and label ( Figure 2a), between the base and linker ( Figure 2b), and may be attached only to the sugar (Figure 2e) or only to the linker ( Figure 2 c).
• the terminator molecule may also contain multiple linkers and moieties which are alternatively spaced together ( Figure 2d).
• the moiety may be attached only to the base, it is believed that the presence of a charged moiety at this position may affect adversely affect the reactivity of the terminator molecule.
• the moiety may also be made of a number of charged units covalently linked together.
• the charge-modified nucleic acid terminators which along with the corresponding nucleic acid terminator decomposition products, migrate on separation media at different rates than the sequencing reaction products and result in improved sequence data (i.e., no blobs which obscure true data) and permit direct loading of nucleic acid sequencing reactions onto separating media.
• the charge-modified nucleic acid terminators work especially well in sequencing reactions which also contain polymerases such as ThermoSequenase, which is Taq ⁇ 271/ F272M/F667Y.
• the foil length enzyme was truncated to eliminate 5' to 3' exonuclease activity, and to provide a polypeptide more stable to proteolysis and heat treatment. Therefore position 1 (amino acid Met) in ThermoSequenase corresponds to position 272 in foil length Taq polymerase.
• Preferred polymerases include ThermoSequenase in which an amino acid substitution has been introduced at E410 (the numbering is for ThermoSequenase, not for Taq polymerase).
• An especially preferred polymerase is ThermoSequenase containing an E410R, E410W, or E41 Q M substitutions.
• Such polymerases are described in PCT/US00/221 0, the entire disclosure of which is incorporated herein by reference.
• Additional preferred polymerases include a foil length version of Taq polymerase with the following substitutions : D18A/E681R F667Y.
• the D18A substitution removes the 5' to 3' exonuclease activity, rather than the deletion of amino acids as in the ThermoSequenase polypeptide.
• the E681R substitution is the position equivalent to E410R in ThermoSequenase
• F667Y is the equivalent position to F396Y in ThermoSequenase.
• This enzyme also has properties desirable for sequencing with dye terminators.
• the amino acid sequence of Taq D18A/E681R F667Y DNA polymerase are also described in PCT/USOO/22150, the entire disclosure of which is incorporated herein by reference.
• the charge-modified nucleic acid te ⁇ ninators may have other important applications. They may also be useful in the therapeutic field as antiviral agents (anti-HIV and anti-HBV etc) (WO 98 49177) and anticancer agents. Many nucleoside and nucleotide analogues have been developed as antiviral agents. They often act by inhibition of DNA polymerase and/or reverse transcriptase activity by a number of means. A number of nucleoside analogues, such as AZT, dd ddt, D4T, and 3TC are being used alone or in combination of other nucleoside or non-nucleoside analogues as anti-H-V .agents.
• the c_____-ge-mod___ed nucleic acid terminators of the present invention may also have antiviral activities alone or in combination with other compounds. Since combination 4rug therapy is being used more frequently to treat viral infections, having an increased number of compounds available by including compounds of the present invention could enhance the possibility of successful treatments.
• the instantly charge-modified terminators could be transported into a cell in either the positively charged state, or if negatively charged, in a dephosphorylated state (which would then convert to a pho ⁇ horylated state ia the cell) or the phosphate groups could be masked to facilitate entry into a cell and the masking groups later removed.
• a charge-modified terminator according to the instant disclosure which may be used in antiviral applications is
• Rhod 5-R110, 5-ROX, 5-TAMRA, 5-REG
• Attachment of the rhodamine dye was carried out using 5-rhodamine hydroxysuccinimde active esters in DMSO N,N-diisopropylethylamine. All the double dye cassettes were purified by reverse phase HPLC prior to conjugation to alkylamino ddNTPs using published methods (and as disclosed in methods disclosed in U.S. Provisional Application No. 60/098,469 filed on August 31, 1998, the entire disclosure of which is hereby incorporated by reference herein.). The labeled ddNTPs were purified by silica gel chromatography followed by ion exchange chromatography then reverse phase HPLC.
• UV/visible spectra were recorded on a Perkin Elmer Lambda 20 UV/visible spectrophotometer in conjunction with WinlabTM software.
• Prep HPLC was carried out on a Waters LC 2000 or LC 4000 system on a C18 Deltapak 15 ⁇ m C18 100A 50x300mm column.
• Ion exchange chromatography was carried out on a Waters LC 600 system.
• the product (retention time 37 min.) was evaporated to dryness in vacua then coevaporated with MeOH (3xl0ml) before lyophilization (lOOmg, 65%).
• UV/vis (1M triethylammonium bicarbonate pH 8.8) 495nm (24670), 465nm (shoulder, 9634), 312nm (6708).
• Figure 3 provides an example of the increase in migration rate relative to sequence products of unincorporated bis-sulfofluorescein energy transfer terminators (and thermal breakdown products thereof) compared to the migration rate of the regular ET terminators.
• fluorescein was attached to -sulfo- ⁇ -alanine to form 5.
• a portion of 5 was attached to a second ⁇ -sulfo- ⁇ -alanine moiety to form 6 which was subsequently attached to 11-ddCTP to form 8,
• a control ddNTP containing regular FAM attached to 11-ddCTP was also synthesized. The structures were run in a single color sequencing reaction to determine the effect of the charge on mobility.
• compound 7 As fluorescein carries a net 1- charge, compound 7 is considered as overall 2- linker arm, compound 8 has an overall 3- linker arm eharge.
• N-(N-5-carboxamidofluorescein- ⁇ -sulfo- ⁇ -alanine)a ⁇ nido- ⁇ -sulfo- ⁇ -a1anine (6.
• N-5-carboxam_dofluorescein- ⁇ -sulfo- ⁇ -akni__e (5, 50mg, 0.095mmol) was dissolved in DMF (3ml) thenN ⁇ diisopropylethylamine (0.25ml, 15eq.) and TSTU (42mg, 1.5eq.) added. The reaction mixture was stirred at room temperature for Ih. then ⁇ -sulfo- ⁇ -alanine (24mg, l_5eq.) added.
• the modified dye (lmmol) was dissolved in DMF ⁇ 5ml) then disuccinimdyl carbonate (4eq.) and DMAP (4eq.) were added at -60°C.
• the reaction mixture was stirred at -30°C for 15 min. then a solution of aminoalkyl-ddNTP (0.67eq., Na 2 CC> 3 NaHCO 3 pH 8.5) added.
• the reaction was stirred at room temperature for lh. then applied directly to a SiO 2 gel column.
• FIG. 4 illustrates how the net negative charge of the dye labeled dideoxynucleotides effects their (and thermal breakdown products thereof) migration rate. As the net negative charge of the terminator increases, the migration rates of the various peaks seen (each of the peaks seen are either dye labeled dideoxynucleotides or thermal breakdown products thereof) increases (figure 4). At an overall 3- charge (2- from linker, 1-from fluorescein) peaks are absent from the region of the electropherogram where sequence data would normally be obtained.
• a labeled terminator with a 3- charge on the linker arm was synthesized, this time containing an extended linker arm of 18 and 25 atoms.
• Compound 6 was attached to 18-ddCTP and 25-ddCTP using the standard protocol for attachment of labels to ddNTPs outlined in section 2.3. The method of purification was the same for 9 and 10.
• Retention time of 9 Mono-Q ion exchange (47min)
• Retention time of 10 Mono-Q ion exchange (42min)
• Rhod A OH Rhod N OH so 3 - so,- H S0 3 - H S0 3 -
• Rhod rhodamine label
• X length of linker arm
• N base
• the labeled triphosphates 14, 18, 22 were used in direct load sequencing experiment.
• Compound 14 in a direct load experiment showed no breakdown products and with TSII and TaqERDAFY.
• Compounds 18 and 22 gave very dark sequencing bands and were observed to be forming an unexpected aggregate (as observed in the emission spectrum).
• the compounds also produced large colored blobs on a sequencing gel which interfered with interpretation of the sequence.
• Rhod 5R6G
• the starting material was synthesized according to the ET terminator patent and both ⁇ and ⁇ phenylalanine can be used in this chemistry.
• the single dye amino acid was reaeted with ⁇ - sulfo- ⁇ -alanine in DMF/DMSO to yield a compound of formal two minus charge.
• the yield of compound 26 was improved by adding ⁇ -sulfo- ⁇ -alanine as a solution in DMSO.
• Compound 26 was isolated by ion exchange chromatography and the previous reaction repeated to yield compound 27.
• the product was separated from starting material by ion exchange chromatography then the boc group removed by treatment with neat trifluoroacetie acid to yield compound 28.
• the rhodamine dye was introduced by reaction of the amine in aqueous buffer with a DMF solution of the rhodamine active ester.
• the products were isolated by ion exchange chromatography with any unreacted amine 28 recycled from the ion exchange column.
• the charged energy transfer molecules were conjugated to aminoalkynylnucleoside triphosphates using the DSC/DMAP method developed for energy transfer terminators and the products purified by silica gel chromatography, ion exchange separation then finally C18 RP HPLC.
• Boc-J_ phenylal_u_ine-4-(propargyl_ mido(bis-pivaloyl)-5-fluorescein) (400mg, 0.47mmol) was -dissolved in DMF ⁇ 4ml) then DIPEA ⁇ 0.25a_l, 3eq.) added followed by O-(N- succi_ ⁇ im yl)-N,N-N,N-tetr_____£thyleneuroni ⁇ un tetrafluoroborate ⁇ 155mg, l.leq.) in DMF (1ml).
• Boc- ⁇ -phenylalanine-4-(propargylarmdo-5-fluorescein)- ⁇ -suffo- ⁇ -alanine (lOOmg, 0.12 mmol) was dissolved in DMF (3ml) then DIPEA (0.1ml, 3eq.) added followed by O-(N- succinimidyl)-N,N,N,N'-tetramethyleneuroniumtetrafluoroborate (50rng, l.leq.) in DMF (1ml). The reaction was stirred at room temperature for 5min then a solution of ⁇ -sulfo- ⁇ - alanine (25mg, 1.2eq.) in DMSO (1ml, dissolved by gentle heating) added at room temperature.
• N-( Boc- ⁇ -phenylalanine-4- ropargylamido-5-fluorescein)- ⁇ -suffo- ⁇ -_danine)amido- ⁇ - sulfo- ⁇ -alanine (lOOmg, O.lOmmol) was treated with trifluoroacetie acid (10ml) for lh. then the reaction evaporated to dryness in vacuo. The residue was triturated with Et 2 O (30ml), the mother liquor was decanted from the yellow solid which was then dried under high vacuum.
• N-( ⁇ -pheny_aJar_i__e-4- ⁇ propargylamido-5-flu ⁇ alanine (20-0 ⁇ mol) was dissolved in NaHCO 3 /Na 2 CO 3 buffer (0.1M, pH 8.5, 3ml) then the desired rhodamine-NHS active ester (1.5 eq.) in DMF (3ml) added.
• the reaction mixture was stirred at room ten ⁇ erature for 16h, then evaporated to dryness in vacuo.
• the Rl 1 , analog was treated with triethylammonium bicarbonate solution (0.1M, 50ml) for 16h to remove the trifluoroacetimido protecting groups then the product purified by ion exchange chromatography.
• reaction rHx ures were applied directly to a Q-sepharose ion exchange column and eluted with the following gradient.
• A 0.1M TEAB / 40% MeCN (v/v)
• Products eluted in 90% buffer B.
• Unreacted compound 28 eluted from the column in 100% buffer B which could be recycled in later reactions.
• the charged ET cassette (16.0 ⁇ mol) was dissolved in DMF (1ml) then disuecinimdyl carbonate (8 eq.) added as a solid at room temperature.
• the reaction was cooled to -60°C then DMAP (4 eq) in DMF (0.5ml) added.
• the reaction mixture was warmed to -30°C then a solution of aminoalkynl-ddNTP (0.67eq., Na 2 CO 3 /NaHCO3 pH 8.5) added.
• the reaction was stirred at room temperature for lh. then applied directly to a SiO 2 gel column.
• the electropherogram shown in Figure 10 was obtained when compound 40 was used in a sequencing reaction.
• the +2 charged terminator was used in a sequencing reaction and loaded directly on to a slab gel.
• the same experiment was repeated, however the reaction mixture was treated with phosphatase prior to loading on a gel to remove phosphates from the unincorporated dye-labeled dideoxynucleotides remaining in the reaction mixture. This would leave all terminator derived products with an overall positive charge causing them to migrate in the opposite direction to the sequence products during electrophoresis. It is clear from the electropherogram that the colored by-products are absent from the sequence when phosphatase is used to break down the terminator products.
• the rhodamine dye R6G is attached to e-N,N,N-trimethyllysine which contains a formalized positive charge from the ⁇ quaternary amine.
• the product (41) can be further modified to yield a +2 linker arm (42) by reaction with a further molecule of the eharged amino acid. Further reaetion(s) would generate the desired eharged structure.
• the initial molecule studied was lysine, whieh has a number of advantages.
• the pKa of the ⁇ -NH 2 function is approximately 10 hence the amino group will be eharged at the pH f a typical DNA sequencing separation
• TAMRA labeled lysine oUgomers were synthesized and conjugated to 11-ddATP to determine the following information
• oligolysines were selectively protected at the ⁇ -amino group using the desired stoichiometric amount of ethyl trifluoroacetate at 4°C
• the major product was isolated by CIS RP-HPLC and confirmed to be the esired product by electrospray mass spectrometry.
• the free amino group was labeled using 6-TAMRA-NHS active ester then the product -attached to 11-ddATP ris n stan d conjugation chemistry.
• Compounds 51, 52 and 53 were then used in a single color sequencing reaction and the products applied directly to a -sequencing gel ⁇ ABD 377).
• Ohgolysine (O.l ⁇ mmol) was dissolved in MeOH (8ml) then triethylamine (133 ⁇ l, 6eq.) added and the reaction cooled to 4°C.
• Ethyl trifluoroacetate (3eq for 46, 5eq for 47) was added and the reaction stirred at 4°C for 16h.
• the reaction was evaporated to dryness in vacua and the product purified by C18 RPHPLC using the gradient shown below.
• the product containing fractions were evaporated to dryness in vacuo and the product precipitated by the addition of Et_O (50ml).
• the mother liquor was decanted and the solid dried under high vacuum.
• TAMRA labeled protected lysines 48, 49, 50, 3.9 ⁇ mol
• DMF 3ml
• N,N-diisopropylethylamine 0.3ml, 4QQeq.
• O-(N-succinimidyl)- N,N ⁇ r,N-tetramethyleneuronium tetrafluoroborate lOmg, lOeq.
• reaction was stirred at room temperature for lh. then further O-(N- succma ⁇ __dyl)-N,N,N,N ⁇ etra___sthyle_i ⁇ uro tetrafluoroborate (8_ag, 0.4eq.) added.
• the reaction was stirred at room temperature for a further 3Qmin then the solution added to hexa- ⁇ e-trifluoraeeta__t_do)lysine (77mg, 1.0eq_).
• the reaction was stirred at room temperature for 16h. then the product isolated by C18 HPLC eluting with the gradient as shown below. The product containing fractions were evaporated to near dryness in vacuo, then the suspension frozen and lyophilized (31mg, 30%).
• Product eluted at t 50 min.
• TOF MS ES+ found 2023.0 (M+Na+), theoretical C 8 6H 101 F 18 N 1 O 2 2 2023.7.
• reaction mixture was stirred at room temperature for 5 min. then cooled to 0 ⁇ C before addition of 11-ddATP (0.3ml, 4mg, leq) in NaHCO 3 /Na 2 CO 3 (pH 8.5) buffer.
• 11-ddATP 0.3ml, 4mg, leq
• NaHCO 3 /Na 2 CO 3 pH 8.5
• the reaction was raised to room temperature and stirred for a further lh.
• the product was purified by Q sepharose ion exchange chromatography using the gradient shown below;
• the product containing fractions were evaporated to dryness in vacuo and the residure treated with NHjOH (c. 10ml) for 16h.
• the reaction mixture was evaporated to near dryness in vacuo then the product repurified by Q sepharose ion exchange chromatography using the same gradient.
• the product containing fractions were evaporated to dryness in vacuo then dissolved in TE buffer prior to use in a sequencing reaction.
• FAMROX-Lys 6 -l 1-ddCTP was synthesized using the following pathway:
• Compound 59 was attached to 11-ddCTP using O- -succi ⁇ _irnidyl)-N,N,N,N- tetramethyleneuronium tetrafluoroborate as the activating reagent.
• the product was deprotected as a crude mixture using neat trifluoroacetie acid for 20 min. then any unreacted 11-ddCTP removed from the reaction by CIS HPLC.
• the modified nucleotide was then dissolved in DMSO and reacted with a DMSO solution of 5-ROX-NHS active ester.
• H-Lys 6 -11-ddCTP was synthesized as follows:

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