3′-OH unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing
The present invention relates generally to 3′-OH unblocked nucleotides and nucleosides labeled and unlabeled 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 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 method comprising:
exposing a compound, a biological component, or a non-biological component to a light source to remove a photocleavable moiety of the formula:
wherein:
R 3 is alkyl (C3-8) ;
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; and
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≤6) , 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) , arenediyl (C≤12) , heteroarenediyl (C≤12) , or a substituted version of any of these groups;
Y is —O—, —NH—, alkanediyl (C≤12) or substituted alkanediyl (C≤12) ;
n is an integer from 0-6; and
m is an integer from 0-6; or
a -linker-reporter.
2. The method of claim 1 , wherein about 85% to about 100% of the photocleavable moieties are removed by exposure to the light source.
3. The method of claim 1 , wherein the light source emits UV light.
4. The method of claim 3 , wherein the UV light comprises 365 nm wavelength light.
5. The method of claim 1 , wherein the compound, the biological component, or the non-biological component is a nucleobase, a nucleoside, a nucleotide, or a nucleic acid molecule, each of which modified with the photocleavable moiety.
6. The method of claim 1 , wherein a product of the photocleavage step comprises a hydroxymethyl nucleoside, hydroxymethyl nucleotide or a hydroxymethyl nucleotide incorporated into a nucleic acid molecule.
7. The method of claim 1 , wherein the compound, the biological component, or the non-biological component is a protein that has been modified with the photocleavable moiety.
8. The method of claim 1 , wherein R 3 is iso-propyl.
9. The method of claim 1 , wherein R 3 is tert-butyl.
10. The method of claim 1 , wherein R 4 is hydrogen.
11. The method of claim 1 , wherein R 5 is hydrogen, iodo, or methoxy.
12. The method of claim 1 , wherein R 5 is a group of formula:
wherein
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.
13. The method of claim 12 , wherein X is —C≡C— and n is zero.
14. The method of claim 1 , wherein R 5 is a -linker-reporter.
15. The method of claim 14 , wherein the linker is:
wherein
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.
16. The method of claim 15 , wherein X is —C≡C— and n is zero.
17. The method of claim 14 , wherein the reporter is based on a dye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.
18. The method of claim 14 , wherein the reporter is:
19. The method of claim 1 , wherein R 6 is hydrogen.
20. A product prepared by a process comprising the method of claim 1 , wherein the product is derived from the photocleavable moiety and comprises a nitroso group and a ketone group.
21. A compound of the formula:
wherein:
R 3 is alkyl (C3-8) ;
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; and
R 5 and R 6 are each independently:
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; or
alkyl (C≤6 ), alkenyl (C≤6) , alkynyl (C≤6) , aryl (C≤6) , aralkyl (C≤6) , 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.
22. The compound of claim 21 , wherein R 3 is alkyl (C3-4) .
23. The compound of claim 22 , wherein R 3 is iso-propyl.
24. The compound of claim 22 , wherein R 3 is tert-butyl.
25. The compound of claim 22 , wherein R 4 is hydrogen.
26. The compound of claim 22 , wherein R 5 is hydrogen, iodo, or methoxy.