3′-OH unblocked, nucleotides and nucleosides, base modified with photocleavable, terminating groups and methods for their use in DNA sequencing
Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a photocleavable terminating group. The photocleavable-fluorescent group is designed to terminate DNA synthesis as well as be cleaved so that DNA oligomers can be sequenced efficiently in a parallel format. The design of such rapidly cleavable fluorescent groups on nucleotides and nucleosides can enhance the speed and accuracy of sequencing of large oligomers of DNA in parallel, to allow rapid whole genome sequencing, and the identification of polymorphisms and other valuable genetic information, as well as allowing further manipulation and analysis of nucleic acid molecules in their native state following cleavage of the fluorescent group.
1. A compound selected from the group consisting of:
wherein R 1 ═H, monophosphate, diphosphate or triphosphate,
R 2 ═H or OH,
R 3 and R 4 are each independently selected from the group consisting of H, a C 1 -C 12 straight chain or branched alkyl, a C 2 -C 12 straight chain or branched alkenyl or polyenyl, a C 2 -C 12 straight chain or branched alkynyl or polyalkynyl, and an aromatic group, with the proviso that at least one of R 3 and R 4 is H,
R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of H, OCH 3 , NO 2 , CN, a halide, a C 1 -C 12 straight chain or branched alkyl, a C 2 -C 12 straight chain or branched alkenyl or polyenyl, a C 2 -C 12 straight chain or branched alkynyl or polyalkynyl, and an aromatic group, with the proviso that one of R 5 , R 6 , R 7 , and R 8 is a linker group of the structure —C≡CCH 2 NH 2 , —C≡CCH 2 NHC(O)(CH 2 ) 5 NH 2 ,
wherein X═CH 2 , CH═CH, C≡C, O, S, or NH,
Y=CH 2 , O, or NH,
n=an integer from 0-12;
m=an integer from 0-12, and
Dye=a fluorophore.
2. A compound according to claim 1 , wherein R 3 and R 4 are each independently selected from the group consisting of H, —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , isopropyl, tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl, with the proviso that at least one of R 3 and R 4 is H.
3. A compound according to claim 1 , wherein R 3 or R 4 are each independently selected from the group consisting of H, alkyl and aromatic groups optionally containing at least one heteroatom in the alkyl or aromatic groups, and further wherein the aromatic group may optionally be an aryl or polycyclic group, with the proviso that at least one of R 3 and R 4 is H.
4. The compound of claim 2 , wherein R 3 or R 4 is —CH 3 or isopropyl.
5. The compound of claim 1 , further defined as:
6. The compound of claim 5 , further defined as:
7. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
8. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
9. The compound of claim 1 , further defined as:
10. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
11. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
12. The compound of claim 1 , further defined as:
13. The compound of claim 1 , further defined as:
14. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
15. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
16. The compound of claim 1 , further defined as:
or a stereoisomer thereof.
17. The compound of claim 1 , further defined as:
18. The compound of claim 1 , further defined as:
19. The compound of claim 1 , further defined as a compound of formula (I).
20. The compound of claim 1 , further defined as a compound of formula (II).
21. The compound of claim 1 , further defined as a compound of formula (III).
22. The compound of claim 1 , further defined as a compound of formula (IV).
23. The compound of claim 1 , further defined as a compound of formula (V).
24. The compound of claim 1 , further defined as a compound of formula (VI).
25. The compound of claim 1 , further defined as a compound of formula (VII).
26. A compound according to any one of claims 19 - 25 , wherein R 3 and R 4 are each independently selected from the group consisting of H, —CH 3 , —CH 2 CH 3 , —CH 2 CH 2 CH 3 , isopropyl, tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl, with the proviso that at least one of R 3 and R 4 is H.
27. A compound according to any one of claims 19 - 25 , wherein R 3 or R 4 are each independently selected from the group consisting of H, alkyl and aromatic groups optionally containing at least one heteroatom in the alkyl or aromatic groups, and further wherein the aromatic group may optionally be an aryl or polycyclic group, with the proviso that at least one of R 3 and R 4 is H.
28. A compound according to any one of claims 19 - 25 , wherein R 3 or R 4 is —CH 3 or isopropyl.