3′-OH unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing

1. A method of identifying a plurality of bases in a target nucleic acid comprising the following steps:

(i) attaching the 5′-end of a primer to a solid surface;

(ii) hybridizing a target nucleic acid to the primer attached to the solid surface to form a hybridized primer/target nucleic acid complex;

(iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters:

wherein:

R 1 is triphosphate or α-thiotriphosphate;

R 2 is hydrogen or hydroxy;

R 3 is alkyl (C≦8) or substituted alkyl (C≦8) ;

R 4 is

hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;

alkyl (C≦6) , acyl (C≦6) , alkoxy (C≦6) , acyloxy (C≦6) , alkylamino (C≦6) , dialkylamino (C≦6) , amido (C≦6) , or a substituted version of any of these groups;

R 5 is a -linker-reporter, wherein the reporter is based on a dye;

R 6 is:

hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;

alkyl (C≦6) , alkenyl (C≦6) , alkynyl (C≦6) , aryl (C≦6) , aralkyl (C≦8) , heteroaryl (C≦6) , acyl (C≦6) , alkoxy (C≦6) , acyloxy (C≦6) , alkylamino (C≦6) , dialkylamino (C≦6) , amido (C≦6) , or a substituted version of any of these groups;

or a salt, tautomer, or optical isomer thereof;

(iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase reaction;

(v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its reporter;

(vi) exposing the solid surface with the growing primer strand to a light source to remove a photocleavable terminating moiety of the formula:

with the variables as defined by the claim referenced in step (iii); and

(vii) repeating steps (iv) through (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle.

2. The method of claim 1 , wherein step (vi) is conducted in the presence of sodium acetate.

3. The method of claim 1 , wherein steps (v) or (vi) is conducted in the presence of thiourea.

4. The method of claim 1 , wherein step (vi) is conducted in the presence of dithiothreitol (DTT).

5. A method of identifying a plurality of bases in a target nucleic acid comprising the following steps:

(i) attaching the 5′-end of a target nucleic acid to a solid surface;

(ii) hybridizing a primer to the nucleic acid attached to the solid surface to form a hybridized primer/target nucleic acid complex;

(iii) obtaining a polymerase and a compound of formulas I-VII, with the proviso that compounds of different formulas I-VII have different reporters:

wherein:

R 1 is triphosphate or α-thiotriphosphate;

R 2 is hydrogen or hydroxy;

R 3 is alkyl(8) or substituted alkyl(8);

R 4 is

hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;

alkyl (C≦6) , acyl (C≦6) , alkoxy (C≦6) , acyloxy (C≦6) , alkylamino (C≦6) , dialkylamino (C≦6) , amido (C≦6) , or a substituted version of any of these groups;

R 5 is a -linker-reporter, wherein the reporter is based on a dye;

R 6 is:

hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;

alkyl (C≦6) , alkenyl (C≦6) , alkynyl (C≦6) , aryl (C≦6) , aralkyl (C≦8) , heteroaryl (C≦6) , acyl (C≦6) , alkoxy (C≦6) , acyloxy (C≦6) , alkylamino (C≦6) , dialkylamino (C≦6) , amido (C≦6) , or a substituted version of any of these groups;

or a salt, tautomer, or optical isomer thereof;

or a salt, tautomer, or optical isomer thereof;

(iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase reaction;

(v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its reporter;

(vi) exposing the solid surface with the growing primer strand to a light source to remove a photocleavable terminating moiety of the formula:

with the variables as defined by the claim referenced in step (iii); and

(vii) repeating steps (iv) through (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle.

6. The method of claim 5 , wherein step (vi) is conducted in the presence of dithiothreitol (DTT).

7. The method of claim 1 , wherein the incorporation of at least one compound according to step (iv) occurs at about 70% to about 100% of the efficiency of incorporation of its natural nucleotide counterpart.

8. The method of claim 7 , wherein the incorporation efficiency occurs at about 85% to about 100%.

9. The method of claim 1 , wherein the polymerase is selected from the group consisting of reverse transcriptase, terminal transferase, and DNA polymerase.

10. The method of claim 1 , wherein about 85% to about 100% of the photocleavable terminating moieties are removed by exposure to a light source in step (vi).

11. The method of claim 1 , wherein incorporation of at least one compound according to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to about 100%.

12. The method of claim 1 , further comprising prior to step (iv) capping any primers or growing primer strands that did not react in step (iv).

13. The method of claim 1 , further comprising sequencing multiple target nucleic acids synchronistically.

14. The method of claim 1 , wherein R 1 is a triphosphate.

15. The method of claim 1 , wherein R 1 is an α-thiotriphosphate.

16. The method of claim 1 , wherein R 3 is alkyl (C≦8) .

17. The method of claim 16 , wherein R 3 is alkyl (C3-4) .

18. The method of claim 17 , wherein R 3 is tert-butyl.

19. The method of claim 1 , wherein R 4 is hydrogen.

20. The method of claim 1 , wherein the linker of the -linker-reporter is:

wherein

X is

—O—, —S—, or —NH—; or

alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups; and

n is an integer from 0-6.

21. The method of claim 20 , wherein X is —C≡C—.

22. The method of claim 20 , wherein n is zero.

23. The method of claim 1 , wherein the dye is a zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.

24. The method of claim 1 , wherein the reporter is:

25. The method of claim 1 , wherein R 6 is hydrogen.

26. The method of claim 1 , wherein the starred carbon atom is in the S configuration.