Methods of labeling polynucleotides with energy transfer dyes
View Patent ↗Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.
1. A method of forming a fluorescently labeled polynucleotide, comprising the step of enzymatically extending a primer in the presence of a template polynucleotide and a mixture of nucleotides comprising nucleotides suitable for effecting template-dependent primer extension, wherein at least one nucleotide of the mixture is labeled with an energy transfer dye which comprises:
(i) a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;
(ii) a 4,7-dichlororhodamine acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response thereto; and
(iii) a non-nucleosidic linker linking the xanthene donor dye to the 4,7-dichlororhodamine acceptor dye.
2. The method of claim 1 in which the mixture of nucleotides further comprises at least one terminating nucleotide.
3. The method of claim 2 in which the at least one terminating nucleotide is a 2′,3′-dideoxyribonucleotide-5-triphosphate.
4. The method of claim 3 in which the at least one terminating nucleotide is selected from ddATP, ddCTP, ddGTP and ddTTP.
5. The method of claim 2 in which the terminating nucleotide is labeled with the energy transfer dye.
6. The method of claim 1 in which the mixture of nucleotides further comprises four different terminating nucleotides, each of which terminates primer extension at a different template nucleotide.
7. The method of claim 6 in which each terminating nucleotide is labeled with a different, spectrally resolvable fluorescent dye, at least one of which is the energy transfer dye.
8. The method of claim 7 in which each different terminating nucleotide is labeled with an energy transfer dye, a different, spectrally resolvable energy transfer dye per terminating nucleotide.
9. The method of claim 8 in which each energy transfer dye comprises the same donor dye.
10. The method of claim 7 in which each terminating nucleotide is a 2′,3′-dideoxyribonucleotide-5′-triphosphate.
11. The method of claim 7 in which the terminating nucleotides comprise a mixture of ddATP, ddCTP, ddGTP and ddTTP.
12. The method of claim 1 in which the nucleotides comprise a mixture of dATP, dCTP, dGTP and dTTP.
13. The method of claim 1 in which the non-nucleosidic linker links the xanthene donor dye to the 4′-, 5- or 6-position of the 4,7-dichlororhodamine acceptor dye.
14. The method of claim 1 in which the non-nucleosidic linker has a backbone that is less than 9 atoms in length.
15. The method of claim 1 in which the linker comprises a functional group selected from an alkene, a diene, an alkyne, a five membered ring having at least one unsaturated bond, a six membered ring having at least one unsaturated bond and a fused ring structure.
16. The method of claim 1 in which the xanthene donor dye is a fluorescein dye.
17. The method of claim 16 in which the non-nucleosidic linker links the 4′-, 5- or 6-position of the fluorescein donor dye to the 4′-, 5- or 6-position of the 4,7-dichlororhodamine dye.
18. The method of claim 17 in which energy transfer dye is covalently linked to the base of the nucleotide via the 4′-, 5- or 6-position of the fluorescein donor dye or the 4′-position of the 4,7-dichlororhodamine acceptor dye.
19. The method of claim 18 in which energy transfer dye is covalently linked to the base of the nucleotide by way of an acetylenic amido or alkenic amido linkage.
20. The method of claim 19 in which the acetylenic amido or alkenic amido linkage is selected from —C≡C—CH 2 —NH—C(O)—, 3-amino-1-propyn-1-yl, —C≡C—CH 2 —NH—C(O)—(CH 2 ) 5 —C(O)—, —C═CH—C(O)—NH—(CH 2 ) 5 —NH—C(O)— and —C≡C—CH 2 —O—CH 2 —CH 2 —NR—, where R is hydrogen, a protecting group or alkyl.
21. The method of claim 1 in which the 4,7-dichlororhodamine acceptor dye comprises structure (I):
wherein:
R 1 , R 2 , R 3 and R 4 are each, independently of one another, selected from hydrogen and alkyl, or alternatively R 1 and R 5 , R 2 and R 6 , R 3 and R 8 and/or R 4 and R 9 are taken together with the atoms to which they are bonded to form a 5, 6 or 7-membered ring;
R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 6 and R 7 and/or R 9 and R 10 are taken together with the atoms to which they are bonded to form a benzo group;
X 1 , X 3 and X 4 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy; and
R 8 , X 3 or X 4 comprises the non-nucleosidic linker linking the xanthene donor dye.
22. The method of claim 1 in which the xanthene donor dye comprises structure (II):
wherein:
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 12 and R 13 and/or R 15 and R 16 are taken together with the atoms to which they are bonded to form a benzo group;
X 11 , X 12 , X 13 , X 14 and X 15 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy; and
R 14 , X 13 or X 14 comprises the non-nucleosidic linker linking the 4,7-dichlororhodamine acceptor dye.
23. The method of claim 1 in which energy transfer dye comprises structure (III):
wherein:
R 1 , R 2 , R 3 and R 4 are each, independently of one another, selected from hydrogen, methyl and ethyl, or alternatively R 1 and R 5 , R 2 and R 6 , R 3 and R 5 and/or R 4 and R 9 are taken together with the atoms to which they are bonded to form a 5, 6 or 7-membered ring;
R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 6 and R 7 and/or R 9 and R 10 are taken together with the atoms to which they are bonded to form a benzo group;
X 1 and X 3 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy;
L is the non-nucleosidic linker;
R 11 , R 12 , R 13 , R 14 , R 15 and R 16 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 12 and R 13 and/or R 15 and R 16 are taken together with the atoms to which they are bonded to form a benzo group; and
X 11 , X 12 , X 13 and X 15 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy,
with the proviso that one of R 8 or R 14 comprises a linkage that covalently links the energy transfer dye to the nucleotide.
24. The method of claim 1 in which energy transfer dye comprises structure (IV):
wherein:
R 1 , R 2 , R 3 and R 4 are each, independently of one another, selected from hydrogen, methyl and ethyl, or alternatively R 1 and R 5 , R 2 and R 6 , R 3 and R 8 and/or R 4 and R 9 are taken together with the atoms to which they are bonded to form a 5, 6 or 7-membered ring;
R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 6 and R 7 and/or R 9 and R 10 are taken together with the atoms to which they are bonded to form a benzo group;
X 1 and X 3 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy;
L is the non-nucleosidic linker;
R 11 , R 12 , R 13 , R 15 and R 16 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile, alkoxy, phenyl and substituted phenyl, or alternatively, R 12 and R 13 and/or R 15 and R 16 are taken together with the atoms to which they are bonded to form a benzo group; and
X 11 , X 12 , X 13 , X 14 and X 15 are each, independently of one another, selected from hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, sulfone, amino, ammonium, amido, nitrile and alkoxy,
with the proviso that one of R 8 , X 13 or X 14 comprises a linkage that covalently links the energy transfer dye to the nucleotide.
25. The method of claim 23 or claim 24 in which X 1 is carboxylate.
26. The method of claim 25 in which R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and X 3 are each hydrogen.
27. The method of claim 25 in which R 1 , R 3 , R 5 , R 7 , R 8 , R 10 and X 3 are each hydrogen; R 2 and R 4 are each ethyl; and R 6 and R 9 are each methyl.
28. The method of claim 25 in which R 1 , R 2 , R 3 and R 4 are each methyl; and R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and X 3 are each hydrogen.
29. The method of claim 25 in which R 1 and R 5 , R 2 and R 6 , R 3 and R 3 and R 4 and R 9 are taken together with the atoms to which they are bonded to form six-membered rings; and R 7 , R 10 and X 3 are each hydrogen.
30. The method of claim 25 in which R 1 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , R 10 and X 3 are each hydrogen; R 2 is ethyl; and R 6 is methyl.
31. The method of claim 25 in which R 1 , R 5 , R 7 , R 8 , R 9 , R 10 and X 3 are each hydrogen; R 3 , R 4 and R 6 are each methyl; and R 2 is ethyl.
32. The method of claim 25 in which R 5 , R 6 , R 7 , R 10 and X 3 are each hydrogen; R 1 and R 2 are each methyl; and R 3 and R 8 and R 4 and R 9 are taken together with the atoms to which they are bonded to form six-membered rings.
33. The method of claim 25 in which R 1 , R 2 , R 5 , R 6 , R 7 , R 10 and X 3 are each hydrogen; and R 3 and R 8 and R 4 and R 9 are taken together with the atoms to which they are bonded to form six-membered rings.
34. The method of claim 25 in which R 1 , R 5 , R 6 , R 7 , R 10 and X 3 are each hydrogen; R 2 is ethyl; and R 3 and R 8 and R 4 and R 9 are taken together with the atoms to which they are bonded to form six-membered rings.
35. The method of claim 25 in which R 1 , R 2 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 and X 3 are each hydrogen; and R 3 and R 4 are each methyl.