POLYMERASE ENZYME FROM PYROCOCCUS ABYSSI

Abstract

The present invention relates to a polymerase enzyme with improved ability to incorporate reversibly terminating nucleotides. The enzyme comprising the following mutations in the motif A region (SGS). It relates to a polymerase enzyme according to SEQ ID NO. 1 with mutations in the amino acid sequence positions 409, 410 and 411.

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Claims

1. A polymerase enzyme according to SEQ ID NO. 1 or any polymerase that shares at least 70%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity thereto, comprising the following mutation(s): a. at position 409 of SEQ ID NO. 1: i. serine (S) (L409S) or, ii. glutamine (Q) (L409Q) or, iii. tyrosine (Y) (L409Y) or, iv. phenylalanine (F) (L409F) b. at position 410 of SEQ ID NO. 1: i. glycine (G) (Y410G) or, ii. adenine (A) (Y409A) or, iii. serine (S) (Y409S), c. at position 411 of SEQ ID NO. 1: i. serine (S) (P411S) or, ii. isoleucine (I) (P411I) or, iii. cysteine (C) (P411C) or, iv. adenine (A) (P411A), wherein the enzyme has little or no 3′-5′ exonuclease activity.

2. The polymerase enzyme of claim 1, wherein the polymerase is from an organism belonging to the family of Thermococcaceae, preferably from the genera of Pyrococcus.

3. The polymerase enzyme according to claim 1, wherein the polymerase enzyme comprises a L409S mutation, a Y410G mutation and a P411S mutation; and optionally comprises one or more a D141A mutation, a E143A mutation, or a A486L mutation.

4. The polymerase enzyme according to claim 3, wherein the polymerase enzyme further comprises the A486L mutation.

5. The polymerase enzyme according to claim 1, wherein the polymerase enzyme shares 95% or 98% sequence identity with SEQ ID NO. 1 and comprises the following mutations: (i) L409S, Y410G, P411S and (ii) A486L.

6. The polymerase enzyme according to claim 1, wherein the polymerase enzyme exhibits an increased rate of incorporation of nucleotides which have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group, compared to the control polymerase.

7. A nucleic acid molecule encoding a polymerase enzyme according to claim 1.

8. An expression vector comprising the nucleic acid molecule of claim 7.

9. A method for incorporating nucleotides which have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group into DNA comprising the following substances (i) a polymerase enzyme according to claim 1, (ii) template DNA, (iii) one or more nucleotides, which have been modified at the 3′ sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′ hydroxyl group.

10. Use of a polymerase enzyme according to claim 1 for DNA sequencing, DNA labeling, primer extension, amplification or the like.

11. A kit comprising a polymerase enzyme according to claim 1.

Description

FIGURE CAPTIONS

[0092] FIG. 1 shows labeled analogs of nucleotides with 3′-O-methylenedisulfide-containing protecting group, where labels are attached to the nucleobase via cleavable oxymethylenedisulfide linker (—OCH2-SS—). The analogs are (clockwise from the top left) for deoxyadenosine, thymidine or deoxyuridine, deoxycytidine and deoxyguanosine.

[0093] FIG. 2 shows an example of the labeled nucleotides where the spacer of the cleavable linker includes the propargyl ether linker. The analogs are (clockwise from the top left) for deoxyadenosine, thymidine or deoxyuridine, deoxycytidine and deoxyguanosine.

[0094] FIG. 3 shows a synthetic route of the labeled nucleotides specific for labeled dT intermediate.

[0095] FIG. 4 shows a cleavable linker synthesis starting from an 1,4-butanediol.

[0096] FIG. 5 shows the measurement of polymerase performance using extension in solution and capillary electrophoresis. The rate of single base terminating dNTP incorporation is measured. The extended fluorescent primer is detected by capillary electrophoresis (CE). The relative rate of dNTP addition is determined by plots of fraction extended primer over time.

[0097] FIG. 6. shows the sequencing performance of Jpol 104 (P. Abyssi construct, SEQ ID No 2) as measured by sequencing KPIs and compared to legacy (T9).

[0098] FIG. 7. shows example reads generated by JPol 104 (SEQ ID #2) and T9 polymerases using GR sequencer. The bar chart shows example read as final intensities vs cycle number. The intensities were subjected to background, crosstalk and phasing correction. Color coding is as follows: C—blue, T—green, A—yellow, G—red. For each cycle there is one dominating color indicative of base nucleotide incorporated and base called.

[0099] FIG. 8 shows kinetics of incorporation of nucleotide analogs (reversibly terminating dG) as measured by capillary electrophoresis assay. The methodology used here is solution based assay using synthetic DNA template and synthetic primer labeled with fluorophore at 5′end. The template is specific to the nucleotide interrogated. A mixture of pre-annealed primer/template, polymerase and nucleotide are incubated at temperature appropriate for the polymerase studied. After incubation an aliquot is loaded onto capillary electrophoresis system where size separation is performed using denaturing conditions and fluorescence detection. Peaks corresponding to non-extended primer, extended primer and residual nuclease activity (primer degradation) are observed in this trace indicating polymerase ability to incorporate nucleotide analog.

[0100] FIG. 9 shows generic universal building blocks structures comprising new cleavable linkers usable with the enzymes of the present invention. PG=Protective Group, LI, L2—linkers (aliphatic, aromatic, mixed polarity straight chain or branched). RG=Reactive Group. In one embodiment of present invention such building blocks carry an Fmoc protective group on one end of the linker and reactive NHS carbonate or carbamate on the other end. This preferred combination is particularly useful in modified nucleotides synthesis comprising new cleavable linkers. A protective group should be removable under conditions compatible with nucleic acid/nucleotides chemistry and the reactive group should be selective. After reaction of the active NHS group on the linker with amine terminating nucleotide, an Fmoc group can be easily removed using base such as piperidine or ammonia, therefore exposing amine group at the terminal end of the linker for the attachment of cleavable marker. A library of compounds comprising variety of markers can be constructed this way very quickly.

[0101] FIG. 10 shows activity of several enzymes of the present invention with either 3′-O—CH.sub.2N.sub.3 or 3′-O—CH.sub.2SSCH.sub.3 terminating groups as measured by capillary electrophoresis assay. Activity is expressed as fraction of extended template over specific time.

[0102] FIG. 11 shows incorporation of fluorescently labeled, reversibly terminating nucleotide Alexa488-dC-3′-O—CH.sub.2SSCH.sub.3 as measured by fluorescence plate based assay for polymerases of the present invention: JPo1104 (SEQ ID #2), JPo1127 (SEQ ID #3), JPo1128 (SEQ ID #4), JPo1129 (SEQ ID #5). Duplex DNA was immobilized on the plate, a solution of polymerase and nucleotide was added and after incubation plate was washed and read with fluorescence plate reader (exc. 490 nm/em. 520 nm).

EXAMPLES

[0103]

TABLE-US-00002 Enzyme Sequences SEQ ID NO.1 MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKD gi|1495740|emb| DSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYL CAA90888.1| EHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEG Wild type NEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKS DNA-dependent IDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPYL DNA polymerase LKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLFP Pyrococcus abyssi AIRRTINLPTYTLETVYEVIFGKSKEKVYAHEIAEAWETGKGL ERVAKYSMEDAKVTSELGKEFFPMEAQLARLVGHPVWDVSR SSTGNLVEWFLLTKAYERNELAPNKPDEREYERRLRESYEGG YVNEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNRENCKEY DVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKDP VEKKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVT AWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIKE KALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEEG KIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIVK EVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLAA KGVKVKPGMVIGYIVLXGDGPISKRAIAIEEFDPKKHKYDAE YYIENQVLPAVERILRAFGYRKEDLRYQKTKQVGLGAWLKF SEQ ID NO. 2 MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKD >JPol104_abyssi DSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYL (SGS) EHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEG NEELTFLAVAIATLYHEGEEFGKGPIIMISYADEEGAKVITWK SIDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPY LLKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLF PAIRRTINLPTYTLETVYEVIFGKSKEKVYAHEIAEAWETGKG LERVAKYSMEDAKVTSELGKEFFPMEAQLARLVGHPVWDVS RSSTGNLVEWFLLTKAYERNELAPNKPDEREYERRLRESYEG GYVNEPEKGLWEGIVSLDFRSSGSSIIITHNVSPDTLNRENCKE YDVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKD PVEKKLLDYRQRLIKILANSYYGYYGYAKARWYCKECAESV TAWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIK EKALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEE GKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIV KEVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLA AKGVKVKPGMVIGYIVLRGDGPISKRAIAIEEFDPKKHKYDAE YYIENQVLPAVERILRAFGYRKEDLRYQKTKQVGLGAWLKF SEQ ID NO. 3 MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKD JPol127_QAI_ DSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYL abyssi EHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEG (QAI) NEELTFLAVAIATLYHEGEEFGKGPIIMISYADEEGAKVITWK SIDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPY LLKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLF PAIRRTINLPTYTLETVYEVIFGKSKEKVYAHEIAEAWETGKG LERVAKYSMEDAKVTSELGKEFFPMEAQLARLVGHPVWDVS RSSTGNLVEWFLLTKAYERNELAPNKPDEREYERRLRESYEG GYVNEPEKGLWEGIVSLDFRSQAISIIITHNVSPDTLNRENCKE YDVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKD PVEKKLLDYRQRLIKILANSYYGYYGYAKARWYCKECAESV TAWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIK EKALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEE GKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIV KEVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLA AKGVKVKPGMVIGYIVLRGDGPISKRAIAIEEFDPKKHKYDAE YYIENQVLPAVERILRAFGYRKEDLRYQKTKQVGLGAWLKFS GS SEQ ID NO. 4 MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKD JPol128_YSC_ DSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYL abyssi EHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEG (YSC) NEELTFLAVAIATLYHEGEEFGKGPIIMISYADEEGAKVITWK SIDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPY LLKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLF PAIRRTINLPTYTLETVYEVIFGKSKEKVYAHEIAEAWETGKG LERVAKYSMEDAKVTSELGKEFFPMEAQLARLVGHPVWDVS RSSTGNLVEWFLLTKAYERNELAPNKPDEREYERRLRESYEG GYVNEPEKGLWEGIVSLDFRSYSCSIIITHNVSPDTLNRENCKE YDVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKD PVEKKLLDYRQRLIKILANSYYGYYGYAKARWYCKECAESV TAWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIK EKALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEE GKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIV KEVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLA AKGVKVKPGMVIGYIVLRGDGPISKRAIAIEEFDPKKHKYDAE YYIENQVLPAVERILRAFGYRKEDLRYQKTKQVGLGAWLKFS GS SEQ ID NO. 5 MIIDADYITEDGKPIIRIFKKEKGEFKVEYDRTFRPYIYALLKD JPol129_FSA_ DSAIDEVKKITAERHGKIVRITEVEKVQKKFLGRPIEVWKLYL abyssi EHPQDVPAIREKIREHPAVVDIFEYDIPFAKRYLIDKGLTPMEG (FSA) NEELTFLAVAIATLYHEGEEFGKGPIIMISYADEEGAKVITWK SIDLPYVEVVSSEREMIKRLVKVIREKDPDVIITYNGDNFDFPY LLKRAEKLGIKLPLGRDNSEPKMQRMGDSLAVEIKGRIHFDLF PAIRRTINLPTYTLETVYEVIFGKSKEKVYAHEIAEAWETGKG LERVAKYSMEDAKVTSELGKEFFPMEAQLARLVGHPVWDVS RSSTGNLVEWFLLTKAYERNELAPNKPDEREYERRLRESYEG GYVNEPEKGLWEGIVSLDFRSFSASIIITHNVSPDTLNRENCKE YDVAPQVGHRFCKDFPGFIPSLLGNLLEERQKIKKRMKESKD PVEKKLLDYRQRLIKILANSYYGYYGYAKARWYCKECAESV TAWGRQYIDLVRRELESRGFKVLYIDTDGLYATIPGAKHEEIK EKALKFVEYINSKLPGLLELEYEGFYARGFFVTKKKYALIDEE GKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVDEAVKIV KEVTEKLSKYEIPPEKLVIYEQITRPLSEYKAIGPHVAVAKRLA AKGVKVKPGMVIGYIVLRGDGPISKRAIAIEEFDPKKHKYDAE YYIENQVLPAVERILRAFGYRKEDLRYQKTKQVGLGAWLKFS GS

Example 1

Synthesis of 3′-O-(methylthiomethyl)-5′-0-(tert-butyldimethylsilyl)-2′-deoxythymidine (2)

[0104] 5′-0-(tert-butyldimethylsilyl)-2′-deoxythymidine (1) (2.0 g, 5.6 mmol) was dissolved in a mixture consisting of DMSO (10.5 mL), acetic acid (4.8 mL), and acetic anhydride (15.4 mL) in a 250 mL round bottom flask, and stirred for 48 hours at room temperature. The mixture was then quenched by adding saturated K.sub.2CO.sub.3 solution until evolution of gaseous CO2 was stopped. The mixture was then extracted with EtOAc (3×100 mL) using a separating funnel. The combined organic extract was then washed with a saturated solution of NaHCO.sub.3 (2×150 mL) in a partitioning funnel, and the organic layer was dried over Na.sub.2S0.sub.4. The organic part was concentrated by rotary evaporation. The reaction mixture was finally purified by silica gel column chromatography.

Example 2

Synthesis of 3′-O-(ethyldithiomethyl)-2′-deoxythymidine (4)

[0105] Compound 2 (1.75 g, 4.08 mmol), dried overnight under high vacuum, dissolved in 20 mL dry CH.sub.2Cl.sub.2 was added with EtsN (0.54 mL, 3.87 mmol) and 5.0 g molecular sieve-3A, and stirred for 30 min under Ar atmosphere. The reaction flask was then placed on an ice-bath to bring the temperature to sub-zero, and slowly added with 1.8 eq 1M SO.sub.2CI.sub.2 in CH2CI2 (1.8 mL) and stirred at the same temperature for 1.0 hour. Then the ice-bath was removed to bring the flask to room temperature, and added with a solution of potassium thiotosylate (1.5 g) in 4 mL dry DMF and stirred for 0.5 hour at room temperature.

[0106] Then 2 eq EtSH (0.6 mL) was added and stirred additional 40 min. The mixture was then diluted with 50 mL CH.sub.2Cl.sub.2 and filtered through celite-S in a funnel. The sample was washed with adequate amount of CH.sub.2Cl.sub.2 to make sure that the product was filtered out. The CH.sub.2Cl.sub.2 extract was then concentrated and purified by chromatography on a silica gel column (Hex:EtOAC/1:1 to 1:3, Rf=0.3 in Hex:EtOAc/1:1). The resulting crude product was then treated with 2.2 g of NH.sub.4F in 20 mL MeOH. After 36 hours, the reaction was quenched with 20 mL saturated NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2 by partitioning. The CH.sub.2Cl.sub.2 part was dried over Na.sub.2SO.sub.4 and purified by chromatography (Hex:EtOAc/1:1 to 1:2).

Example 3

Synthesis of the triphosphate of 3′-O-(ethyldithiomethyl)-2′-deoxythymidine (5)

[0107] In a 25 mL flask, compound 4 (0.268 g, 0.769 mmol) was added with proton sponge (210 mg), equipped with rubber septum. The sample was dried under high vacuum for overnight. The material was then dissolved in 2.6 mL (MeO).sub.3PO under argon atmosphere. The flask, equipped with Ar-gas supply, was then placed on an ice-bath, stirred to bring the temperature to sub-zero. Then 1.5 equivalents of POCI.sub.3 was added at once by a syringe and stirred at the same temperature for 2 hours under Argon atmosphere. Then the ice-bath was removed and a mixture consisting of tributylammonium-pyrophosphate (1.6 g) and Bu.sub.3N (1.45 mL) in dry DMF (6 mL) was prepared. The entire mixture was added at once and stirred for 10 min. The reaction mixture was then diluted with TEAB buffer (30 mL, 100 mM) and stirred for additional 3 hours at room temperature. The crude product was concentrated by rotary evaporation, and purified by CI 8 Prep HPLC (method: 0 to 5 min 100% A followed by gradient up to 50% B over 72 min, A=50 mM TEAB and B=acetonitrile). After freeze drying of the target fractions, the semi-pure product was further purified by ion exchange HPLC using PL-SAX Prep column (Method: 0 to 5 min 100% A, then gradient up to 70% B over 70 min, where A=15% acetonitrile in water, B=0.85M TEAB buffer in 15% acetonitrile). Final purification was carried out by CI8 Prep HPLC as described above resulting in ˜25% yield of compound 5.

Example 4

Synthesis of N.SUP.4.-Benzoyl-5′-0-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-2 ‘ deoxycytidine (7)

[0108] N.sup.4-benzoyl-5′-O-(tert-butyldimethylsilyl)-2’-deoxycytidine (6) (50 g, 112.2 mmol) was dissolved in DMSO (210 mL) in a 2 L round bottom flask. It was added sequentially with acetic acid (210 mL) and acetic anhydride (96 mL), and stirred for 48 h at room temperature. During this period of time, a complete conversion to product was observed by TLC (Rf=0.6, EtOAc:hex/10:1 for the product).

[0109] The mixture was separated into two equal fractions, and each was transferred to a 2000 mL beaker and neutralized by slowly adding saturated K.sub.2CO.sub.3 solution until CO.sub.2 gas evolution was stopped (pH 8). The mixture was then extracted with EtOAc in a separating funnel. The organic part was then washed with saturated solution of NaHCO.sub.3 (2×1 L) followed by with distilled water (2×1 L), then the organic part was dried over Na.sub.2SO.sub.4.

[0110] The organic part was then concentrated by rotary evaporation. The product was then purified by silica gel flash-column chromatography using puriflash column (Hex:EtOAc/1:4 to 1:9, 3 column runs, on 15 um, HC 300 g puriflash column) to obtain N.sup.4-benzoyl-5′-O-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-2′-deoxycytidine (7) as grey powder in 60% yield.

Example 5

N.SUP.4.-Benzoyl-3 ‘-0-(ethyldithiomethyl)-5’-0-(tert-butyldimethylsilyl)-2′-deoxycytidine (8)

[0111] N.sup.4-Benzoyl-5′-0-(tert-butyldimethylsilyl)-3′-O-(methylthiomethyl)-2′-deoxycytidine (7) (2.526 g, 5.0 mmol) dissolved in dry CH.sub.2Cl.sub.2 (35 mL) was added with molecular sieve-3A (10 g). The mixture was stirred for 30 minutes. It was then added with Et3N (5.5 mmol), and stirred for 20 minutes on an ice-salt-water bath. It was then added slowly with 1M SO.sub.2CI.sub.2 in CH.sub.2Cl.sub.2 (7.5 mL, 7.5 mmol) using a syringe and stirred at the same temperature for 2 hours under N2-atmosphere. Then benzenethiosulfonic acid sodium salt (1.6 g, 8.0 mmol) in 8 mL dry DMF was added and stirred for 30 minutes at room temperature. Finally, EtSH was added (0.74 mL) and stirred additional 50 minutes at room temperature. The reaction mixture was filtered through celite-S, and washed the product out with CH.sub.2Cl.sub.2. After concentrating the resulting CH.sub.2Cl.sub.2 part, it was purified by flash chromatography using a silica gel column (1:1 to 3:7/Hex:EtOAc) to obtain compound 8 in 54.4% yield.

Example 6

N.SUP.4.-Benzoyl-3′-O-(ethyldithiomethyl)-2′-deoxycytidine (9)

[0112] N.sup.4-Benzoyl-3 ‘-O-(ethyldithiomethyl)-5′-O-(tert-butyldimethylsilyl)-2’-deoxycytidine (8, 1.50 g, 2.72 mmol) was dissolved in 50 mL THF. Then 1M TBAF in THF (3.3 mL) was added at ice-cold temperature under nitrogen atmosphere. The mixture was stirred for 1 hour at room temperature. Then the reaction was quenched by adding 1 mL MeOH, and solvent was removed after 10 minutes by rotary evaporation. The product was purified by silica gel flash chromatography using gradient 1:1 to 1:9/Hex:EtOAc to result in compound 9. Finally, the synthesis of compound 10 was achieved from compound 9 following the standard synthetic protocol described in the synthesis of compound 5.

[0113] The synthesis of the labeled nucleotides can be achieved following the synthetic routes shown in FIG. 3 and FIG. 4. FIG. 3 is specific for the synthesis of labeled dT intermediate, and other analogs could be synthesized similarly.