2-nitrobenzyl-modified ribonucleotides

1. A ribonucleoside having a formula SM-BASE, wherein:

SM is a ribose,

BASE is selected from the group consisting of adenine, guanine, cytosine and uracil,

the ribose comprises a reversible chain terminating moiety at a 2′ position in said ribose,

the reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group, and

the ribonucleoside is labeled with a detectable label.

2. The ribonucleoside of claim 1 , wherein the ribonucleoside is a ribonucleotide selected from the group consisting of ribonucleoside 5′-phosphate, a ribonucleoside 5′-diphosphate and a ribonucleoside 5′-triphosphate.

3. The ribonucleoside of claim 1 , wherein the ribonuceloside is a ribonucleotide selected from the group consisting of adenosine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-triphosphate, guanosine 5′-monophosphate, guanosine 5′-diphosphate, guanosine 5′-triphosphate, uridine 5′-monophosphate, uridine 5′-diphosphate, uridine 5′-triphosphate, cytidine 5′-monophosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate.

4. The ribonucleoside of claim 1 , wherein the reversible chain terminating moiety is connected to the ribose by a reversible linkage.

5. The ribonucleoside of claim 4 , wherein the reversible linkage is bonded to the ribose by a bond that is cleavable by electromagnetic radiation, chemical treatment, or a combination thereof.

6. The ribonucleoside of claim 5 wherein the electromagnetic radiation is light.

7. The ribonucleoside of claim 1 , wherein the reversible chain terminating moiety is a 2-Nitrobenzyl group.

8. The ribonucleoside of claim 1 , wherein the reversible chain terminating moiety is a Desyl group.

9. The ribonucleoside of claim 1 , wherein the reversible chain terminating moiety is a p-hydroxyphenacyl caging group.

10. The ribonucleoside of claim 1 , wherein the detectable label is a moiety is selected from the group consisting of green fluorescent protein, blue fluorescent protein, red fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucoronidase, luciferases, b-lactamase, digoxygenin, fluorescent dye molecule, fluorescein, cy3, cy5, alkaline phosphatase and horse radish peroxidase.

11. The ribonucleoside of claim 1 , wherein the detectable label is removable by photobleaching.

12. The ribonucleoside of claim 1 , wherein the detectable label is connected to the ribonucleoside by a reversible linkage.

13. The ribonucleoside of claim 12 , wherein the reversible linkage is bonded to the ribonucleoside by a bond that is cleavable by electromagnetic radiation, chemical treatment, or a combination thereof.

14. A method, comprising:

(a) providing a ribonucleoside having a formula SM-BASE, wherein SM is a ribose, and BASE is selected from the group consisting of adenine, guanine, cytosine and uracil; and

(b) attaching a reversible chain terminating moiety at a 2′ position of said ribose, and

(c), before or after (b), attaching a detectable label to ribonucleoside,

wherein the reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group.

15. A method comprising:

(a) elongating a primer:target nucleic acid complex with a RNA polymerase and at least one first tpccics of a first ribonucleotide having the formula PM-SM-BASE to form an incorporated ribonucleotide, wherein the primer:target nucleic acid complex comprises a primer complexed with a target nucleic acid, the incorporated ribonucleotide comprises a detectable label, PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, and the ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′ position of the ribose; and

(b) detecting the incorporated ribonucleotide by detecting the detectable label to determine a sequence of the target nucleic acid,

wherein the reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group.

16. The method of claim 15 further comprising of:

(c) removing the chain terminating moiety of the incorporated ribonucleotide by breaking the reversible linkage; and

(d) repeating (a), (b) and (c) with of a second ribonucleotide which is different from the first ribonucleotide, the second ribonucleotide having the formula PM′-SM′-BASE′, wherein PM′ is a phosphate moiety, SM′ is a ribose, BASE′ is a pyrimidine or purine, and the ribose of the second ribonucleotide comprises a chain terminating moiety connected by a reversible linkage at a 2′ position of the ribose of the second ribonucleotide.

17. The method of claim 15 , further comprising forming the primer:target nucleic acid by hybridization or synthesis by RNA polymerase.

18. The method of claim 15 wherein the RNA polymerase is a phage-encoded RNA polymerase selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase and SP6 RNA polymerase.

19. A method comprising:

(a) elongating a primer:target nucleic acid complex with a RNA polymerase and a first ribonucleotide to form an incorporated nucleotide, wherein the primer:target nucleic acid complex comprises a primer complexed with a target nucleic acid, the first ribonucleotide is selected from the group consisting of ATP, GTP, UTP and CTP, the first ribonucleotide comprises a ribose and a chain terminating moiety connected by a reversible linkage at a 2′ position of the ribose, and the incorporated ribonucleotide is reversibly labeled with a detectable label; and

(b) detecting the incorporated nucleotide by detecting the detectable label to determine a sequence of the target nucleic acid.

20. The method of claim 19 , further comprising:

(c) removing said the terminating moiety and the detectable label from the incorporated ribonucleotide; and

(d) repeating (a), (b) and (c) with a second ribonucleotide which is different from the first ribonucleotide.

21. The method of claim 19 , further comprising forming the primer:target nucleic acid by hybridization or synthesis by RNA polymerase.

22. The method of claim 19 or 20 , wherein the RNA polymerase is a phage-encoded RNA polymerase selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase and SP6 RNA polymerase.

23. The method of claim 19 , wherein the RNA polymerase comprises a steric gate deletion which can incorporate a 2′ modified ribonucleotide into an elongating nucleic acid.

24. The method of claim 19 , wherein the RNA polymerase comprises additional amino acid substitutions in a nucleotide binding and catalytic pocket which can incorporate a 2′ modified ribonucleotide into an elongating nucleic acid.

25. The method of claim 14 , wherein the ribonucleoside is a ribonucleotide selected from the group consisting of ribonucleoside 5′-phosphate, a ribonucleoside 5′-diphosphate, and a ribonucleoside 5′-triphosphate.

26. The method of claim 14 , wherein the ribonuceloside is a ribonucleotide selected from the group consisting of adenosine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-triphosphate, guanosine 5′-monophosphate, guanosine 5′-diphosphate, guanosine 5′-triphosphate, uridine 5′-monophosphate, uridine 5′-diphosphate, uridine 5′-triphosphate, cytidine 5′-monophosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate.

27. The method of claim 14 , wherein the reversible chain terminating moiety is a 2-Nitrobenzyl group.

28. The method of claim 14 , wherein the reversible chain terminating moiety is a Desyl group.

29. The method of claim 14 , wherein the reversible chain terminating moiety is a p-hydroxyphenacyl caging group.

30. The method of claim 15 , wherein BASE is selected from the group consisting of adenine, guanine, cytosine and uracil.

31. The method of claim 15 , wherein the first ribonucleotide is selected from the group consisting of ribonucleoside 5′-phosphate, a ribonucleoside 5′-diphosphate, and a ribonucleoside 5′-triphosphate.

32. The method of claim 15 , wherein the first ribonucleotide is selected from the group consisting of adenosine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-triphosphate, guanosine 5′-monophosphate, guanosine 5′-diphosphate, guanosine 5′-triphosphate, uridine 5′-monophosphate, uridine 5′-diphosphate, uridine 5′-triphosphate, cytidine 5′-monophosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate.

33. The method of claim 15 , wherein the reversible chain terminating moiety is a 2-Nitrobenzyl group.

34. The method of claim 15 , wherein the reversible chain terminating moiety is a Desyl group.

35. The method of claim 15 , wherein the reversible chain terminating moiety is a p-hydroxyphenacyl caging group.

36. The method of claim 19 , wherein the first ribonucleotide is selected from the group consisting of ribonucleoside 5′-phosphate, a ribonucleoside 5′-diphosphate, and a ribonucleoside 5′-triphosphate.

37. The method of claim 19 , wherein the first ribonucleotide is selected from the group consisting of adenosine 5′-monophosphate, adenosine 5′-diphosphate, adenosine 5′-triphosphate, guanosine 5′-monophosphate, guanosine 5′-diphosphate, guanosine 5′-triphosphate, uridine 5′-monophosphate, uridine 5′-diphosphate, uridine 5′-triphosphate, cytidine 5′-monophosphate, cytidine 5′-diphosphate, and cytidine 5′-triphosphate.

38. The method of claim 19 , wherein the reversible chain terminating moiety is a 2-Nitrobenzyl group.

39. The method of claim 19 , wherein the reversible chain terminating moiety is a Desyl group.

40. The method of claim 19 , wherein the reversible chain terminating moiety is a p-hydroxyphenacyl caging group.