Dye-labeled ribonucleotide triphosphates
View Patent ↗The invention provides novel dye-labeled ribonucleotide analogs and methods for synthesizing those analogs. The compounds of the invention are especially useful for DNA sequencing by the polymerase chain reaction.
1. A compound having the formula:
wherein B is a nucleobase; L is a linker selected from a propargyl-ethyl-oxide-amino linker, a propargyl linker, a benzylamine linker, a rigid linker, a tuned linker, and a heterocycle linker; R 3 is triphosphate, α-thiotriphosphate, or a salt thereof, and Dye is a fluorescein-type dye, a rhodamine-type dye, an energy transfer dye pair, or a cyanine-type dye.
2. The compound according to claim 1 , wherein the rhodamine-type dye is a 4,7-dichlorophenyl-rhodamine dye.
3. The compound according to claim 2 , wherein the 4,7-dichlorophenyl-rhodamine dye is selected from dTAMRA, dROX, dR6G, and dR110.
4. The compound according to claim 1 , wherein the nucleobase is selected from cytosine, adenine, uracil, guanine, 7-deazaadenine, and 7-deazaguanine.
5. A compound of the formula II:
wherein L is a linker selected from a propargyl-ethyl-oxide-amino linker, a propargyl linker, a propargyl-propyl-oxide-amino linker, a benzylamine linker, a rigid linker, and a multimer thereof;
wherein R 4 is either NH 2 , OH, or O, and R 5 is either NH 2 , OH, or H;
wherein R 3 is either triphosphate, α-thiotriphosphate, or a salt thereof; and
wherein the dye is any reporter group.
6. A compound having the formula:
wherein B is a nucleobase selected from uracil, cytosine, adenine, 7-deazaadenine, guanine, and 7-deazaguanine;
wherein R 3 is triphosphate or a salt thereof;
wherein L is a linker selected from propargyl-ethyl-oxide-amino and propargyl wherein the linker is attached to the 8-C of a adenine, 7-deazaadenine, guanine, or 7-deazaguanine nucleobase, the 7-C or 8-C of a 7-deazaadenine or 7-deazaguanine nucleobase, or the C-5 of a uracil or cytosine nucleobase; and
wherein Dye is selected from a rhodamine-type dye and a fluorescein-type dye.
7. The compound according to claim 6 , wherein B is uracil; L is propargyl-ethyl-oxide-amino or propargyl; and Dye is TAMRA or dTAMRA.
8. The compound according to claim 6 , wherein B is cytosine; L is propargyl-ethyl-oxide-amino or propargyl; and Dye is ROX or dROX.
9. The compound according to claim 6 , wherein B is 7-deazaadenine; L is propargyl-ethyl-oxide-amino or propargyl; and Dye is R6G or dR6G.
10. The compound according to claim 6 , wherein B is 7-deazaguanine; L is propargyl-ethyl-oxide-amino or propargylamino; and Dye is R110 or dR110.
11. The compound according to claim 6 , wherein Dye is an energy transfer dye pair comprised of a fluorescein-type dye and a rhodamine-type dye wherein the fluorescein-type dye is attached to the nucleobase through a propargyl-ethyl-oxide-amino or propargyl linker, and the fluorescein-type dye is attached to the rhodamine-type dye by a second linker.
12. The compound according to claim 11 , wherein the fluorescein-type dye is 4-aminomethyl FAM and the rhodamine-type dye is a 4,7-dichlororhodamine dye.
13. The compound according to claim 12 , wherein the 4,7-dichlororhodamine dye is dTAMRA, dROX, dR6G, or dR110.