Labelled nucleotides
The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).
1. A labeled nucleoside triphosphate having the structure:
wherein Z is a removable protecting group;
X is a triphosphate group;
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine;
Linker comprises
wherein the double asterisks ** indicates the attachment point to the nucleobase; and
Label is a fluorophore.
2. The labeled nucleoside triphosphate of claim 1 , wherein Z is azidomethyl.
3. The labeled nucleoside triphosphate of claim 2 , wherein the nucleobase is selected from the group consisting of
wherein the asterisk * indicates the attachment point to the sugar moiety of the nucleoside triphosphate.
4. The labeled nucleoside triphosphate of claim 3 , wherein the Label comprises a cyanine, a rhodamine, or a coumarin dye.
5. The labeled nucleoside triphosphate of claim 3 , wherein the nucleobase is
and the Label is a rhodamine dye.
6. The labeled nucleoside triphosphate of claim 3 , wherein the nucleobase is
and the Label is a Cy5 dye.
7. The labeled nucleoside triphosphate of claim 3 , wherein the nucleobase is
and the Label is a rhodamine dye.
8. The labeled nucleoside triphosphate of claim 3 , having the structure:
or a salt thereof.
9. The labeled nucleoside triphosphate of claim 3 , where the Linker is cleavable by a water soluble phosphine.
10. The labeled nucleoside triphosphate of claim 1 , wherein Z comprises an azido group.
11. A labeled nucleoside triphosphate having the structure:
wherein Z is a removable protecting group;
X is a triphosphate group;
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine;
Linker comprises
and
Label is a fluorophore.
12. The labeled nucleoside triphosphate of claim 11 , wherein the Linker further comprises a propargyl amine or propargyl amide moiety.
13. The labeled nucleoside triphosphate of claim 11 , wherein Z comprises an azido group.
14. The labeled nucleoside triphosphate of claim 11 , wherein Z is azidomethyl.
15. The labeled nucleoside triphosphate of claim 14 , wherein the nucleobase is selected from the group consisting of
wherein the asterisk * indicates the attachment point to the sugar moiety of the nucleoside triphosphate.
16. The labeled nucleoside triphosphate of claim 15 , wherein the Label comprises a cyanine, a rhodamine, or a coumarin dye.
17. The labeled nucleoside triphosphate of claim 15 , wherein the nucleobase is
and the Label is a rhodamine dye.
18. The labeled nucleoside triphosphate of claim 15 , wherein the nucleobase is
and the Label is a Cy5 dye.
19. The labeled nucleoside triphosphate of claim 15 , wherein the nucleobase is
and the Label is a rhodamine dye.
20. The labeled nucleoside triphosphate of claim 15 , where the Linker is cleavable by a water soluble phosphine.
21. A labeled nucleoside triphosphate having the structure:
wherein Z is a removable protecting group comprising an azido group;
X is a triphosphate group;
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine;
Linker comprises an aryl ring having the structure
a moiety of the structure
and a propargyl amine or propargyl amide moiety, wherein X is O, and wherein *** indicates where the moiety is connected to the remainder of the nucleoside triphosphate; and
Label is a fluorophore.
22. The labeled nucleoside triphosphate of claim 21 , wherein Z is azidomethyl.
23. The labeled nucleoside triphosphate of claim 21 , wherein the nucleobase is selected from the group consisting of
wherein the asterisk * indicates the attachment point to the sugar moiety of the nucleoside triphosphate.
24. The labeled nucleoside triphosphate of claim 23 , wherein the nucleobase is
and the Label is a rhodamine dye.
25. The labeled nucleoside triphosphate of claim 23 , wherein the nucleobase is
and the Label is a Cy5 dye.
26. The labeled nucleoside triphosphate of claim 23 , wherein the nucleobase is
and the Label is a rhodamine dye.
27. The labeled nucleoside triphosphate of claim 21 , where the Linker is cleavable by a water soluble phosphine.
28. The labeled nucleoside triphosphate of claim 27 , wherein the Linker is cleavable and the Z is removal under the same condition using the water soluble phosphine.
29. The labeled nucleoside triphosphate of claim 21 , wherein the Linker further comprises one or more spacer units comprising —O—, —NH—, or —N(alkyl)-.