Reversibly blocked nucleoside analogues and their use

1. A nucleoside analogue of the following formula:

wherein

R 1 is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, cyanoethenaldehyde oximyl, cis-cyanoethenyl, trans-cyanoethenyl, cis-cyanofluoroethenyl, trans-cyanofluoroethenyl, biscyanoethenyl, bisfluoroethenyl, cis-propenyl, trans-propenyl, nitroethenyl, acetoethenyl, methylcarbonoethenyl, amidoethenyl, methylsulfonoethenyl, methylsulfonoethyl, formimidate, formhydroxymate, vinyloethenyl, ethylenoethenyl, cyanoethylenyl, nitroethylenyl, amidoethylenyl,

wherein R a is hydrogen, cyano, halogen, nitro, alkoxy, substituted or unsubstituted amino, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkenyl; and R b is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted alkoxy; and

wherein R a is hydrogen, cyano, halogen, nitro, alkoxy, substituted or unsubstituted amino, substituted or unsubstituted sulfonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, and substituted or unsubstituted carbonyl;

R 2 comprises a nucleobase; and

R 3 is a cleavable linking moiety comprising at least three phosphates, or analogues thereof.

2. The nucleoside analogue of claim 1 , wherein R 3 comprises a 5′-O-1-thio triphosphate.

3. The nucleoside analogue of claim 1 , wherein the nucleoside analogue has the formula of Formula VIII:

wherein X is O or S and the nucleoside analogue is a substrate for a DNA polymerase; or

wherein the nucleoside analogue has the following formula:

wherein

R 2 consists of the nucleobase;

L is a linker; and

D is a detectable fluorescent label; or

wherein the nucleoside analogue has the following formula:

wherein

X is selected from the group consisting of 0 and S; or

wherein the nucleoside analogue comprises the following formula:

wherein R 2 is the nucleobase and D comprises a rhodamine dye or a derivative thereof; or

wherein the nucleoside analogue comprises the following formula:

4. A nucleoside analogue of Formula VI:

wherein

R 1 is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl;

R 2 is a nucleobase;

L is a linker;

A 1 comprises an affinity tag;

X is selected from the group consisting of O and S; and

the nucleoside analogue is a substrate for a DNA polymerase.

5. The nucleoside analogue of claim 4 , wherein L is a cleavable linker.

6. The nucleoside analogue of claim 5 , wherein R 1 and L can be cleaved from the nucleoside analogue under the same conditions.

7. The nucleoside analogue of claim 4 , wherein A 1 comprises a nitrilotriacetic acid (NTA) affinity tag, a peptide affinity tag comprising at least six contiguous histidine amino acids, an affinity tag selected from the group consisting of biotin, a small molecule antigen, and a peptide.

8. The nucleoside analogue of claim 7 , wherein the small molecule antigen is selected from the group consisting of a fluorophore, an amphetamine, a barbituate, a benzodiazepine, a cocaine metabolite, a cannabinoid, a cannabinoid metabolites, tetrahydrocannabinol, methadone, an opiate, propoxyphene, phencyclidine, digoxigenin, and DNP.

9. The nucleoside analogue of claim 7 , wherein the peptide is selected from the group consisting of a His tag, a Myc tag, a Flag tag, an HA tag, a V5 tag, an AviTag, a calmodulin tag, an E tag, an S tag, an SBP tag, a Softag, a Strep tag, a TC tag, a VSV tag, an Xpress tag, glutathione, an isopeptag, and a SpyTag.

10. The nucleoside analogue of claim 4 , wherein the nucleoside analogue comprises the following formula:

wherein R 2 is the nucleobase.

11. The nucleoside analogue of claim 10 , wherein the nucleobase is selected from the group consisting of a 7-substituted 7-deaza adenine analogue, a 7 substituted 7-deaza guanine analogue, a 5-substituted thymine, and a 5-substituted cytosine.

12. A composition comprising a nucleoside analogue of the following formula:

wherein

R 1 is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl;

X is selected from the group consisting of O and S; and

R 2 comprises a nucleobase, and optionally a linker and a detectable label or affinity agent,

wherein the nucleoside analogue is covalently linked via the 5′ phosphate or thiophosphate to an oligonucleotide.

13. The composition of claim 12 , wherein R 2 comprises a nucleobase, a linker, and a detectable label or affinity agent.

14. A composition comprising

i) a nucleoside analogue of the following formula:

or

ii) a nucleoside analogue of Formula IX:

wherein

R 1 is a reversible blocking group selected from the group consisting of cyanoethenyl, allenyl, formaldehyde oximyl, acrylaldehyde oximyl, propionaldehyde oximyl, and cyanoethenaldehyde oximyl;

X is selected from the group consisting of O and S;

R 2 is a nucleobase;

L is a linker;

A 1 comprises an affinity tag; and

A 2 comprises a detectably labeled affinity agent that forms a specific and non-covalent complex with A 1 , wherein the nucleoside analogue is covalently linked via the 5′ phosphate or thiophosphate to an oligonucleotide.

15. The composition of claim 14 , wherein A 1 comprises a fluorescent dye selected from the group consisting of a fluorone dye, a rhodamine dye, a cyanine dye, a coumarin dye, a phycoerythrin, and an allophycocyanine.

16. A method of sequencing comprising:

i) providing a reaction mixture comprising template nucleic acid, a primer, a polymerase, and a first nucleoside analogue of claim 4 ;

ii) extending the primer by incorporating the first nucleoside analogue with the polymerase;

iii) contacting the incorporated first nucleoside analogue with a detectably labeled affinity agent that forms a specific and non-covalent complex with A 1 of the incorporated first nucleoside analogue, thereby specifically labeling the incorporated first nucleoside analogue; and

iv) detecting the specifically labeled incorporated first nucleoside analogue.

17. A nucleoside analogue of the following formula:

wherein:

R 1 is

wherein R a , R b , and R c are each individually selected from the group consisting of hydrogen, cyano, halogen, nitro, alkoxy, substituted or unsubstituted amino, substituted or unsubstituted sulfonyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, and substituted or unsubstituted carbonyl; and is a double bond;

R 2 comprises a nucleobase; and

R 3 is a cleavable linking moiety comprising at least three phosphates, or analogues thereof.

18. The nucleoside analogue of claim 1 , wherein R 1 is cis-cyanoethenyl or trans-cyanoethenyl.