AFFINITY TAG LABELED NUCLEOSIDES AND USES

1 . A nucleoside analogue of the following formula:

wherein

R 1 is a reversible blocking group selected from the group consisting of azidomethyl, nitrobenzyl, coumarinyl, nitronaphthalenyl, aminoxyl, and carbonyl;

R 2 is a nucleobase;

L is a linker;

A 1 comprises a non-fluorescent affinity tag;

X is selected from the group consisting of O and 5; and

the nucleoside analogue is a substrate for a DNA polymerase.

2 . The nucleoside analogue of claim 1 , wherein L is a cleavable linker.

3 . The nucleoside analogue of claim 2 , wherein R 1 and L can be cleaved from the nucleoside analogue under the same conditions.

4 . The nucleoside analogue of claim 1 , wherein A 1 comprises a non-fluorescent affinity tag selected from the group consisting of nitrilotriacetic acid (NTA) and a peptide comprising at least six contiguous histidine amino acids.

5 . The nucleoside analogue of claim 1 , wherein A 1 comprises a non-fluorescent affinity tag selected from the group consisting of biotin, vitamin D 3 , a non-fluorescent small molecule antigen, and a peptide.

6 . The nucleoside analogue of claim 5 , wherein the non-fluorescent small molecule antigen is selected from the group consisting of an amphetamine, a barbituate, a benzodiazepine, a cocaine metabolite, a cannabinoid, a cannabinoid metabolite, tetrahydrocannabinol, methadone, an opiate, propoxyphene, phencyclidine, digoxigenin, digoxin, and DNP.

7 . The nucleoside analogue of claim 5 , wherein the peptide antigen 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.

8 . The nucleoside analogue of claim 5 , wherein the nucleoside analogue comprises the following formula:

wherein R 2 is the nucleobase.

9 . The nucleoside analogue of claim 8 , 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.

10 . A composition comprising

i) a nucleoside analogue of the following formula:

wherein

R 1 is a reversible blocking group selected from the group consisting of azidomethyl, nitrobenzyl, coumarinyl, nitronaphthalenyl, aminoxyl, and carbonyl;

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

R 2 is a nucleobase;

L is a linker;

A 1 comprises a fluorescent or non-fluorescent 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.

11 . The composition of claim 10 , 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.

12 . A method of sequencing comprising:

i) providing a reaction mixture comprising template nucleic acid, a primer, a polymerase, and a first nucleoside analogue of Formula VII:

wherein

R 1 is a reversible blocking group selected from the group consisting of azidomethyl, nitrobenzyl, coumarinyl, nitronaphthalenyl, aminoxyl, and carbonyl;

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

R 2 is a nucleobase;

L is a linker; and

A comprises a non-fluorescent or fluorescent affinity tag;

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 of the incorporated first nucleoside analogue, thereby specifically labeling the incorporated first nucleoside analogue; and

iv) detecting the specifically labeled incorporated first nucleoside analogue.

13 . The method of claim 12 , wherein the detectably labeled affinity agent is fluorescently labeled, and the detection comprises detecting a fluorescence emission from the fluorescently labeled affinity agent in complex with A of the incorporated first nucleoside analogue.

14 . The method of claim 12 , wherein the method further comprises:

v) cleaving the reversible blocking group R 1 of the incorporated first nucleoside analogue thereby removing the blocking group from the incorporated first nucleoside analogue;

vi) cleaving the linker L thereby removing the non-fluorescent or fluorescent binding molecule A of the incorporated first nucleoside analogue, or quenching the label of the detectably labeled affinity agent in complex with A of the incorporated first nucleoside analogue;

vii) providing a second nucleoside analogue of Formula VII and a polymerase;

viii) extending the primer by incorporating the second nucleoside analogue with the polymerase;

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

x) detecting the specifically labeled incorporated second nucleoside analogue.

15 . A method of sequencing comprising:

i) providing a reaction mixture comprising template nucleic acid, a primer, a ligase, and an oligonucleotide comprising a 5′ portion and a 3′ portion comprising a first nucleoside analogue of Formula X:

wherein

R 1 is a reversible blocking group selected from the group consisting of azidomethyl, nitrobenzyl, coumarinyl, nitronaphthalenyl, aminoxyl, and carbonyl;

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

R 2 is a nucleobase;

L is a linker;

A comprises a non-fluorescent or fluorescent affinity tag; and

denotes a 5′ phosphodiester bond between the nucleoside analogue of Formula X and the 5′ portion of the oligonucleotide;

ii) hybridizing the oligonucleotide comprising the first nucleoside analogue of Formula X to the template nucleic acid at a position 3′ of, and adjacent to, the primer;

iii) ligating the hybridized oligonucleotide to the adjacent primer with the ligase, thereby incorporating the first nucleoside analogue of Formula X into the primer;

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

v) detecting the specifically labeled incorporated first nucleoside analogue.

16 . The method of claim 15 , wherein the detectably labeled affinity agent is fluorescently labeled, and the detection comprises detecting a fluorescence emission from the fluorescently labeled affinity agent in complex with A of the incorporated first nucleoside analogue.

17 . The method of claim 15 , wherein the method further comprises:

vi) cleaving the linker L thereby removing the non-fluorescent or fluorescent binding molecule A of the incorporated first nucleoside analogue, or quenching the label of the detectably labeled affinity agent in complex with A of the incorporated first nucleoside analogue;

vii) cleaving the reversible blocking group R 1 of the hybridized oligonucleotide comprising the first nucleoside analogue of Formula X;

viii) providing a second oligonucleotide, the oligonucleotide comprising a 5′ portion and a 3′ portion, wherein the 3′ portion comprises a second nucleoside analogue of Formula X, and a ligase;

ix) hybridizing the second oligonucleotide comprising the second nucleoside analogue of Formula X to the template nucleic acid at a position 3′ of, and adjacent to, the primer;

x) ligating the hybridized oligonucleotide to the adjacent primer with the ligase, thereby incorporating the second nucleoside analogue of Formula X into the primer;

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

xii) detecting the specifically labeled incorporated second nucleoside analogue.