Energy transfer dyes with enhanced fluorescence
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 fluorescently-labeled reagent comprising a reagent and an energy transfer dye, wherein the energy transfer dye comprises:
a xanthene donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response thereto;
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
a non-nucleosidic linker linking the 5- or 6-ring position of the donor dye to the 5- or 6-ring position of the acceptor dye,
and wherein the energy transfer dye is covalently linked to the reagent.
2. The fluorescently-labeled reagent of claim 1 in which the reagent is selected from the group consisting of proteins, polypeptides, polysaccharides, nucleosides/tides, oligonucleotides, oligonucleotide analogs, lipids, solid supports and organic and inorganic polymers.
3. The fluorescently-labeled reagent of claim 2 in which the reagent is a nucleoside/tide.
4. The fluorescently labeled reagent of claim 3 in which the energy transfer dye is covalently linked to the base of the nucleoside/tide at the 4′-position of the donor or acceptor dye.
5. The fluorescently labeled reagent of claim 4 in which the energy transfer dye is covalently linked to the base of the nucleoside/tide by way of an acetylenic amido or alkenic amido linkage.
6. The fluorescently labeled reagent of claim 5 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≡CH 2 —O—CH 2 —CH 2 —NR—, where R is hydrogen, a protecting group or alkyl.
7. The fluorescently-labeled reagent of claim 3 in which the donor dye is a fluorescein.
8. The fluorescently-labeled reagent of claim 3 in which the linker has a backbone that is less than 9 atoms in length.
9. The fluorescently-labeled reagent of claim 3 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.
10. The fluorescently-labeled reagent of claim 3 in which the energy transfer dye comprises the structure:
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 may be 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 may be 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 linker linking the donor and acceptor dyes;
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 may be taken together with the atoms to which they are bonded to form a benzo group;
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; and
R 8 or R 14 comprises the attachment to the nucleoside/tide.
11. The fluorescently labeled reagent of claim 10 in which the nucleoside/tide is a 2′-deoxyribonucleoside.
12. The fluorescently labeled reagent of claim 10 in which the nucleoside/tide is a 2′-deoxyribonucleotide.
13. The fluorescently labeled reagent of claim 12 in which the 2′-deoxyribonucleotide is a 2′-deoxyribonucleoside-5′-triphosphate.
14. The fluorescently labeled reagent of claim 10 in which the nucleoside/tide is a terminating nucleotide.
15. The fluorescently labeled nucleoside/tide of claim 14 in which the terminating nucleotide is a 2′,3′-dideoxynucleoside-5′-triphosphate.
16. A fluorescently-labeled reagent of claim 2 in which the reagent is an oligonucleotide or oligonucleotide analog.
17. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye is attached to the 5′-terminus of the oligonucleotide or analog.
18. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye is attached to the 3′-terminus of the oligonucleotide or analog.
19. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye is attached to a nucleobase of the oligonucleotide or analog.
20. The fluorescently-labeled reagent of claim 16 in which the energy transfer dye comprises the structure:
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 may be 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 may be 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 linker linking the donor and acceptor dyes;
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 may be taken together with the atoms to which they are bonded to form a benzo group;
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; and
R 8 or R 14 comprises the attachment to the oligonucleotide or analog.