Energy transfer dyes with enchanced fluorescence
First Claim
1. An energy transfer dye having the structure ##STR121## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response;
- ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;
C(O) is a carbonyl group;
Z1 is selected from the group consisting of NH, sulfur and oxygen;
R21 is a C1-5 alkyl attached to the donor dye;
R22 is a substituent selected from the group consisting of 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
R28 includes a functional group which attaches the linker to the acceptor dye.
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Accused Products
Abstract
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 R21 Z1 C(O)R22 R28 where R21 is a C1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z1 is either NH, sulfur or oxygen, R22 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 R28 includes a functional group which attaches the linker to the acceptor dye.
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Citations
79 Claims
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1. An energy transfer dye having the structure ##STR121## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response;
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ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; C(O) is a carbonyl group; Z1 is selected from the group consisting of NH, sulfur and oxygen; R21 is a C1-5 alkyl attached to the donor dye; R22 is a substituent selected from the group consisting of 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 R28 includes a functional group which attaches the linker to the acceptor dye. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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12. An energy transfer dye having the structure ##STR124## where C(O) is a carbonyl group;
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Y1 and Y2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z1 is selected from the group consisting of NH, sulfur and oxygen; R11 -R17 are each independently selected from the group consisting of hydrogen, fluorine, chlorine bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof; R21 is a C1-5 alkyl; R22 is a substituent selected from the group consisting of 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; R28 includes a functional group which attaches the linker to the acceptor dye; and ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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27. An energy transfer fluorescent dye having the general structure ##STR129## wherein:
- Y1, Y1 '"'"', Y2 and Y2 '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,
R11 -R16 and R11 '"'"'-R16 '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof, and X1 -X5 and X1 '"'"'-X5 '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof; Y1, Y2, R11 -R16, and X1 -X5 are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response; Y1 '"'"', Y2 '"'"', R11 '"'"'-R16 '"'"', and X1 '"'"'-X5 '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and one of X3 and X4 and one of X3 '"'"' and X4 '"'"' are taken together to form a linker linking the donor to the acceptor dye such that energy is transferred from the donor to the acceptor dye. - View Dependent Claims (28, 29, 30)
- Y1, Y1 '"'"', Y2 and Y2 '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,
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31. An energy transfer fluorescent dye selected from the group consisting of:
- 5 or 6 carboxy TMR-B-CF, 5 or 6 carboxy TMR-F-CF, 5 or 6 carboxy TMR-P-CF, 5 or 6 carboxy TMR-P-CF, 5 or 6 carboxy TMR-A-CF, 5 or 6 carboxy TMR-D-CF, 5 or 6 carboxy TMR-N-CF, 5 or 6 carboxy ROX-CF, CY5-CF, 5 or 6 carboxy TMR-gly-5AMF and 5 or 6 carboxy TMR-5AMF, 5 or 6 carboxy CF-B-TMR-2, 5 or 6 carboxy CFB-DR110-2, 5 or 6 carboxy CFB-DR6g-2, and 5 or 6 carboxy CFB-DROX-2.
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32. A fluorescently labeled reagent comprising:
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a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye including a dye having the structure ##STR130## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response; ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; C(O) is a carbonyl group; Z1 is selected from the group consisting of NH, sulfur and oxygen; R21 is a C1-5 alkyl attached to the donor dye; R22 is a substituent selected from the group consisting of 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 R28 includes a functional group which attaches the linker to the acceptor dye. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47)
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48. A fluorescently labeled reagent comprising:
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a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye including a dye having the structure ##STR133## where C(O) is a carbonyl group; Y1 and Y2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z1 is selected from the group consisting of NH, sulfur and oxygen; R11 -R17 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof; R21 is a C1-5 alkyl; R22 is a substituent selected from the group consisting of 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; R28 includes a functional group which attaches the linker to the acceptor dye; and ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65)
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66. A fluorescently labeled reagent comprising:
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a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye including a dye having the structure ##STR138## wherein;
Y1, Y1 '"'"', Y2 and Y2 '"'"' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,R11 -R16 and R11 -R16 '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof, and X1 -X5 and X1 '"'"'-X5 '"'"' are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, where adjacent substituents are taken together to form a ring, and combinations thereof; Y1, Y2, R11 -R16, and X1 -X5 are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response; Y1 '"'"', Y2 '"'"', R11 '"'"'-R16 '"'"', and X1 '"'"'-X5 '"'"' are selected to correspond to an acceptor dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and one of X3 and X4 and one of X3 '"'"' and X4 '"'"' are taken together to form a linker linking the donor to the acceptor dye such that energy is transferred from the donor to the acceptor dye. - View Dependent Claims (67, 68, 69)
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70. A fluorescently labeled reagent comprising:
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a reagent selected from the group consisting of a nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, oligonucleotide and oligonucleotide analog, modified to be linked to an energy transfer fluorescent dye; and an energy transfer fluorescent dye attached to the reagent, the energy transfer fluorescent dye being selected from the group consisting of;
5 or 6 carboxy TMR-B-CF, 5 or 6 carboxy TMR-F-CF, 5 or 6 carboxy TMR-P-CF, 5 or 6 carboxy TMR-P-CF, 5 or 6 carboxy TMR-A-CF, 5 or 6 carboxy TMR-D-CF, 5 or 6 carboxy TMR-N-CF, 5 or 6 carboxy ROX-CF, CY5-CF, 5 or 6 carboxy TMR-gly-5AMF and 5 or 6 carboxy TMR-5AMF, 5 or 6 carboxy CF-B-TMR-2, 5 or 6 carboxy CFB-DR110-2, 5 or 6 carboxy CFB-DR6g-2, and 5 or 6 carboxy CFB-DROX-2. - View Dependent Claims (71, 72, 73, 74, 75)
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76. A method for sequencing a nucleic acid sequence comprising:
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forming a mixture of extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer; separating the mixture of extended primers; and determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed; the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and having a 3'"'"' end extendable by a polymerase, and an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye having the structure ##STR139## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response; ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; C(O) is a carbonyl group; Z1 is selected from the group consisting of NH, sulfur and oxygen; R21 is a C1-5 alkyl attached to the donor dye; R22 is a substituent selected from the group consisting of 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 R28 includes a functional group which attaches the linker to the acceptor dye.
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77. A method for sequencing a nucleic acid sequence comprising:
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forming a mixture of extended labeled primers by hybridizing a nucleic acid sequence with a fluorescently labeled oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer; separating the mixture of extended primers; and determining the sequence of the nucleic acid sequence by fluorescently measuring the mixture of extended primers formed; the fluorescently labeled oligonucleotide primer including an oligonucleotide sequence complementary to a portion of the nucleic acid sequence being sequenced and having a 3'"'"' end extendable by a polymerase, and an energy transfer fluorescent dye attached to the oligonucleotide, the energy transfer fluorescent dye having the structure ##STR140## where C(O) is a carbonyl group; Y1 and Y2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z1 is selected from the group consisting of NH, sulfur and oxygen; R11 -R17 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof; R21 is a C1-5 alkyl; R22 is a substituent selected from the group consisting of 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; R28 includes a functional group which attaches the linker to the acceptor dye; and ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes.
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78. A method for sequencing a nucleic acid sequence comprising:
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forming a mixture of extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer; separating the mixture of extended primers; and determining the sequence of the nucleic acid sequence by detecting the fluorescently labeled dideoxynucleotide attached to the separated mixture of extended primers; the fluorescently labeled dideoxynucleoside triphosphate including a dideoxynucleoside triphosphate, and an energy transfer fluorescent dye attached to the dideoxynucleoside triphosphate, the energy transfer dye having the structure ##STR141## where DONOR is a dye capable of absorbing light at a first wavelength and emitting excitation energy in response; ACCEPTOR is dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; C(O) is a carbonyl group; Z1 is selected from the group consisting of NH, sulfur and oxygen; R21 is a C1-5 alkyl attached to the donor dye; R22 is a substituent selected from the group consisting of 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 R28 includes a functional group which attaches the linker to the acceptor dye.
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79. A method for sequencing a nucleic acid sequence comprising:
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forming a mixture of extended primers by hybridizing a nucleic acid sequence with a primer in the presence of deoxynucleoside triphosphates, at least one fluorescently labeled dideoxynucleoside triphosphate and a DNA polymerase, the DNA polymerase extending the primer with the deoxynucleoside triphosphates until a fluorescently labeled dideoxynucleoside triphosphate is incorporated onto the extended primer which terminates extension of the primer; separating the mixture of extended primers; and determining the sequence of the nucleic acid sequence by detecting the fluorescently labeled dideoxynucleotide attached to the separated mixture of extended primers; the fluorescently labeled dideoxynucleoside triphosphate including a dideoxynucleoside triphosphate, and an energy transfer fluorescent dye attached to the dideoxynucleoside triphosphate, the dye having the structure ##STR142## where C(O) is a carbonyl group; Y1 and Y2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine; Z1 is selected from the group consisting of NH, sulfur and oxygen; R11 -R17 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, carboxyl, alkyl, alkene, alkyne, sulfonate, amino, ammonium, amido, nitrile, alkoxy, phenyl, substituted phenyl, where adjacent substituents are taken together to form a ring, and combinations thereof; R21 is a C1-5 alkyl; R22 is a substituent selected from the group consisting of 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; R28 includes a functional group which attaches the linker to the acceptor dye; and ACCEPTOR is dye which is capable of absorbing excitation energy emitted by a member of the xanthene class of dyes.
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Specification