3′-OH unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing
The present invention relates generally to 3′-OH unblocked, labeled and unlabeled nucleotides and nucleosides with 5-methoxy-substituted nitrobenzyl-based photocleavable terminating groups for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds, which may be represented by the following formulas: wherein the variables are defined herein, may be used as reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide.
1. A compound of the formula:
wherein:
R 1 is hydroxy, monophosphate, diphosphate, triphosphate, α-thiotriphosphate or polyphosphate;
R 2 is hydrogen or hydroxy;
R 3 is alkyl (C≦8) or substituted alkyl (C≦8) ;
R 4 is
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
alkyl (C≦6) , acyl (C≦6) , alkoxy (C≦6) , acyloxy (C≦6) , alkylamino (C≦6) , dialkyl-amino (C≦6) , amido (C≦6) , or a substituted version of any of these groups;
R 5 and R 6 are each independently:
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
alkyl (C≦6) , alkenyl (C≦6) , alkynyl (C≦6) , aryl (C≦6) , aralkyl (C≦8) , heteroaryl (C≦6) , acyl (C≦6) , alkoxy (C≦6) , acyloxy (C≦6) , alkylamino (C≦6) , dialkyl-amino (C≦6) , amido (C≦6) , or a substituted version of any of these groups;
a group of formula:
wherein
X is
—O—, —S—, or —NH—; or
alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , or a substituted version of any of these groups;
Y is —O—, —NH—, alkanediyl (C≦12) or substituted alkane-diyl (C≦12) ;
n is an integer from 0-6; and
m is an integer from 0-6; or
a -linker-reporter;
or a salt, tautomer, or optical isomer thereof.
2. The compound of claim 1 , further defined as a compound of formula I.
3. The compound of claim 1 , further defined as a compound of formula II.
4. The compound of claim 1 , further defined as a compound of formula III.
5. The compound of claim 1 , further defined as a compound of formula IV.
6. The compound of claim 1 , further defined as a compound of formula V.
7. The compound of claim 1 , further defined as a compound of formula VI.
8. The compound of claim 1 , further defined as a compound of formula VII.
9. The compound of claim 1 , wherein R 1 is hydroxy.
10. The compound of claim 1 , wherein R 1 is a triphosphate.
11. The compound of claim 1 , wherein R 1 is an α-thiotriphosphate.
12. The compound of claim 1 , wherein R 2 is hydrogen.
13. The compound of claim 1 , wherein R 2 is hydroxy.
14. The compound of claim 1 , wherein R 3 is alkyl (C≦8) .
15. The compound of claim 1 , wherein R 3 is isopropyl.
16. The compound of claim 1 , wherein R 3 is tert-butyl.
17. The compound of claim 1 , wherein R 4 is hydrogen.
18. The compound of claim 1 , wherein R 4 is nitro.
19. The compound of claim 1 , wherein R 5 is hydrogen.
20. The compound of claim 1 , wherein R 5 is iodo.
21. The compound of claim 1 , wherein R 5 is alkoxy (C≦6) .
22. The compound of claim 21 , wherein R 5 is methoxy.
23. The compound of claim 1 , wherein R 5 is a -linker-reporter.
24. The compound of claim 1 , wherein R 5 is a -linker-reporter, wherein the linker is:
wherein for either of the above formulas:
X is
—O—, —S—, or —NH—; or
alkanediyl (C≦12) , alkenediyl (C≦12) , alkynediyl (C≦12) , arenediyl (C≦12) , heteroarenediyl (C≦12) , or a substituted version of any of these groups; and
n is an integer from 0-6.
25. The compound of claim 24 , wherein X is alkynediyl (C2-8) .
26. The compound of claim 25 , wherein X is —C≡C—.
27. The compound of claim 24 , wherein n is zero.
28. The compound of claim 24 , wherein the reporter is:
29. The compound of claim 1 , wherein R 6 is hydrogen.
30. The compound of claim 1 , wherein the starred carbon atom is in the S configuration.
31. The compound of claim 1 , wherein the starred carbon atom is in the R configuration.
32. The compound of claim 1 , further defined as:
or a salt and/or protonated form of any of these formulas.
33. The compound of claim 1 , further defined as:
wherein R is ═O or ═S, or a salt and/or protonated form of any of these formulas.
34. The compound of claim 1 , further defined as:
wherein R is ═O or ═S, or a salt and/or protonated form of any of these formulas.
35. The compound of claim 1 , further defined as:
or a salt and/or protonated form of any of these formulas.