Modified nucleosides and nucleotides and uses thereof
The invention is directed to modified guanine-containing nucleosides and nucleotides and uses thereof. More specifically, the invention relates to modified fluorescently labelled guanine-containing nucleosides and nucleotides which exhibit enhanced fluorophore intensity by virtue of reduced quenching effects.
1. A modified nucleoside, comprising a base attached to a fluorophore through a cleavable linking group, wherein the cleavable linking group comprises a cleavable functionality and a hydrophilic spacer group of 5 to 150 atoms.
2. The modified nucleoside of claim 1 , wherein the base is a modified guanine.
3. The modified nucleoside of claim 1 , wherein the base is guanine.
4. The modified nucleoside of claim 1 , further comprising at least one phosphate group.
5. The modified nucleoside of claim 4 , wherein the modified nucleoside is a modified nucleotide triphosphate.
6. The modified nucleoside of claim 1 , wherein the cleavable functionality comprises azide.
7. The modified nucleoside of claim 6 , wherein the cleavable functionality is cleaved using a phosphine.
8. The modified nucleoside of claim 1 , wherein the cleavable functionality comprises an acetal that has an allyl-cleavable functionality.
9. The modified nucleoside of claim 8 , wherein the acetal is cleaved with a transition metal complex.
10. The modified nucleoside of claim 9 , wherein the transition metal complex comprises palladium.
11. The modified nucleoside of claim 9 , wherein the transition metal complex comprises a transition metal selected from the group consisting of platinum, rhodium, ruthenium, osmium, and iridium.
12. The modified nucleoside of claim 8 , wherein the acetal comprises an allyloxy group.
13. The modified nucleoside of claim 8 , wherein the acetal comprises an allyl moiety selected from the group consisting of an allylamino group, an allylthioether group, an allyl carbamate group, an allylurea group, and an S-allylthiocarbamate group.
14. The modified nucleoside of claim 1 , wherein the cleavable functionality is cleaved by a linker-cleavage method selected from the group consisting of electrophilic cleavage, nucleophilic cleavage, photochemical cleavage, reductive cleavage, oxidative cleavage, safety-catch cleavage, and elimination-mediated cleavage.
15. A kit, comprising the modified nucleoside according to claim 1 .
16. A method for detecting a modified nucleoside that has been incorporated into a polynucleotide, comprising:
(a) incorporating the modified nucleoside of claim 1 into a polynucleotide; and
(b) detecting a fluorescent signal from the modified nucleoside that was incorporated in step (a).
17. The method of claim 16 , further comprising providing a template nucleic acid strand and a partially hybridized nucleic acid strand,
wherein step (a) incorporates into the hybridized strand at least one modified nucleoside that is complementary to a nucleoside at the corresponding position of the template strand, and
wherein step (b) identifies the base of the incorporated modified nucleoside, thereby indicating the identity of the complementary nucleoside of the template strand.
18. The method of claim 16 , wherein step (a) further comprises providing a second modified nucleoside that has a different base and a distinguishable fluorescent signal.
19. A reagent for modifying the base of a nucleoside, comprising a fluorophore and a cleavable linking group, wherein the linking group comprises a cleavable functionality and a hydrophilic spacer group of 5 to 150 atoms.