Propargyl substituted nucleoside compounds and methods
Disclosed, among other things, are compounds having the structure wherein X comprises a bond or a linker, LABEL comprises at least one detectable label, W 1 taken alone is —H or —OH, W 2 is —OH or a non-extendable moiety, W 3 when taken alone is —H or when taken together with W 1 is —CH 2 —O—, and W 4 is OH, monophosphate, diphosphate, or triphosphate. Also disclosed are labeled polynucleotide compounds and methods of use thereof.
1. A method of extending a polynucleotide comprising
forming a hybridization complex between a 3′-extendable polynucleotide and a complementary sequence in a template polynucleotide, and
extending the 3′-extendable polynucleotide in the presence of at least one labeled nucleotide 5′-triphosphate and a template-dependent nucleic acid polymerase under conditions effective to append at least one labeled nucleotide to the 3′-end of the extendable polynucleotide,
wherein said at least one labeled nucleotide 5′-triphosphate comprises a labeled nucleotide 5′-triphosphate having the structure
wherein
X is a bond or a linker having from 1-50 atoms, wherein the atoms are selected from C, H, N, O, S, P and Si,
LABEL is at least one detectable label,
W 1 taken alone is —H or —OH,
W 2 is —OH or a non-extendable moiety selected from the group consisting of —H, azido, amino, fluoro, chloro, and methoxy,
W 3 when taken alone is —H or when taken together with W 1 is —CH 2 —O—, and
W 4 is triphosphoryl; and
wherein said extending is performed in the presence of said labeled nucleotide 5′-triphosphate and four different alternate nucleotide triphosphates, selected from ATP, GTP, UTP, and CTP to extend an RNA chain or selected from dATP, dGTP, dCTP and dTTP to extend a DNA chain.
2. The method of claim 1 , wherein the 3′-extendable polynucleotide formed is at least 10-500 nucleotides in length.
3. The method of claim 1 , wherein W 2 is non-extendable.
4. The method of claim 1 , wherein the polymerase comprises a DNA polymerase.
5. The method of claim 1 , wherein W 1 is H.
6. The method of claim 1 , wherein W 2 is H.
7. The method of claim 1 , wherein W 2 is azido.
8. The method of claim 1 , wherein W 2 is amino.
9. The method of claim 1 , wherein W 2 is fluoro.
10. The method of claim 1 , wherein W 2 is chloro.
11. The method of claim 1 , wherein W 2 is methoxy.
12. The method of claim 1 , wherein W 1 is —OH.
13. The method of claim 1 , wherein W 2 is —OH.
14. The method of claim 1 , wherein LABEL is the detectable label selected from the group consisting of a fluorescent dye, an energy transfer dye, a quencher, and biotin.
15. The method of claim 14 , wherein the fluorescent dye is a fluorescein dye, a rhodamine dye or an energy transfer dye.
16. The method of claim 1 , wherein LABEL is a fluorescent dye selected from the group consisting of a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine or a squaraine.
17. The method of claim 1 , wherein LABEL is an energy transfer dye, said energy transfer dye is a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing light emitted by the donor dye.
18. The method of claim 17 , wherein the donor dye is selected from a fluorescein dye and a rhodamine dye.
19. The method of claim 18 , wherein the acceptor dye is selected from the group consisting of a rhodamine dye, a fluorescein dye, a cyanine dye and a non-fluorescent quencher.