Nucleotide analogs

1 . A labeled nucleotide analog of Formula I:

wherein,

B is selected from the group consisting of a purine, a pyrimidine, and analogs thereof,

R 1 is selected from the group consisting of OH and —O-blocking agent,

R 2 is selected from the group consisting of H and OH,

R 3 is a -carbonyl-R 5 -moiety,

R 4 is selected from the group consisting of H and alkyl,

R 5 is an aliphatic linker,

L is a label,

m, at each occurrence, independently is an integer from 1 to 3, and

n, at each occurrence, independently is an integer from 1 to 18.

2 . The labeled nucleotide analog of claim 1 , wherein B is selected from the group consisting of cytosine, uracil, thymine, adenine, guanine, and analogs thereof.

3 . The labeled nucleotide analog of claim 1 , wherein R 4 is H.

4 . The labeled nucleotide analog of claim 1 , 2 or 3 , wherein R 5 is an alkyl.

5 . The labeled nucleotide analog of claim 1 , wherein n is 1, 2 or 3.

6 . The labeled nucleotide analog of claim 1 , wherein L is an optically detectable label.

7 . The labeled nucleotide analog of claim 6 , wherein said optically detectable label is a fluorescent label.

8 . The labeled nucleotide analog of claim 6 , wherein the optically detectable label is selected from the group consisting of cyanine, rhodamine, fluoroscein, coumarin, BODIPY, alexa and conjugated multi-dyes.

9 . The labeled nucleotide analog of claim 6 , wherein the optically detectable label is Cy3 or Cy5.

10 . The labeled nucleotide analog of claim 1 , wherein R 1 is OH.

11 . The labeled nucleotide of analog claim 1 , wherein R 3 is

12 . A method of sequencing a nucleic acid template comprising:

(a) exposing a nucleic acid template hybridized to a primer having a 3′ end to (i) a polymerase capable of catalyzing nucleotide additions to the primer, and (ii) the nucleotide analog of claim 1 under conditions to permit the polymerase to add the nucleotide analog to the primer;

(b) detecting the nucleotide analog added to the primer in step (a);

(c) removing the label from the nucleotide analog;

(d) repeating steps (a), (b) and (c) thereby to determine the sequence of the template.

13 . The method of claim 12 , wherein step (d) is repeated at least three times.

14 . The method of claim 12 , wherein the nucleotide analog, after step (c), is represented by Formula II,

wherein R 2 , B and n are as defined in claim 1 , and

R 8 is a phosphodiester linkage connecting the nucleotide analog to the primer.

15 . The method of claim 12 , wherein during step (a), the template is immobilized to a solid support.

16 . The method of claim 12 , wherein, in step (c), the label is removed by exposure to a reducing agent.

17 . The method of claim 16 , wherein the reducing agent is tris(2-carboxyethyl)phosphine.

18 . The method of claim 16 , wherein the template is immobilized in an array at a density sufficient to detect and sequence single molecules individually.

19 . A method of removing a label from a labeled base in a nucleic acid comprising:

(a) exposing a base of Formula III

wherein R 4 , R 5 , B, L and n are as defined in claim 1 and R 3 is

to a reducing agent for a time sufficient to produce an unlabelled base of Formula IV

wherein B and n are as defined in claim 1 .

20 . The method of claim 19 , wherein the base is a purine, pyrimidine, or an analog thereof.

21 . The method of claim 19 , wherein the base is selected from the group consisting of cytosine, uracil, thymine, adenine, guanine, and analogs thereof.

22 . The method of claim 19 , wherein the reducing agent is tris(2-carboxylethyl)phosphine.

23 . The method of claim 19 , wherein the base is linked to a sugar selected from the group consisting of ribose, deoxyribose, and analogs thereof.

24 . The method of claim 23 , wherein the base and sugar together are present in a nucleotide in a nucleic acid.

25 . The method of claim 12 , wherein the nucleotide analog, after step (c), is represented by Formula II,

wherein R 2 , B and n are as defined in claim 1 ,

R 8 is a phosphodiester linkage connecting the nucleotide analog to an oligonucleotide, and

R 6 is one or more nucleotide analogs comprising a residual moiety, wherein the residue results from removal of a label.