UV excitable fluorescent energy transfer dyes

Abstract

Novel energy transfer dyes which can be used with shorter wavelength light sources are provided. These dyes include a donor dye with an absorption maxima at a wavelength between about 250 to 450 nm and an acceptor dye which is capable of absorbing energy emitted from the donor dye. One of the energy transfer dyes has a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure and an acceptor dye which is capable of absorbing energy emitted from the donor dye, wherein the donor dye has an absorption maxima between about 250 and 450 nm and the acceptor dye has an emission maxima at a wavelength greater than about 500 nm.

What is claimed is:  
     
         1 . An energy transfer dye comprising: 
 a donor dye having an absorption maxima at a wavelength between about 250 to 450 nm; and    an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.    
     
     
         2 . The energy transfer dye according to    claim 1    wherein the donor dye has an absorption maxima at a wavelength between about 300 and 450 nm.  
     
     
         3 . The energy transfer dye according to    claim 1    wherein the donor dye has an absorption maxima at a wavelength between about 350 and 400 nm.  
     
     
         4 . The energy transfer dye according to    claim 1    wherein the acceptor dye has an emission maxima at a wavelength greater than about 550 nm.  
     
     
         5 . The energy transfer dye according to    claim 1    wherein the acceptor dye has an emission maxima at a wavelength between about 500 and 700 nm.  
     
     
         6 . The energy transfer dye according to    claim 1    wherein the acceptor dye has an emission maxima at a wavelength at least about 150 nm greater than the absorption maxima of the donor dye.  
     
     
         7 . The energy transfer dye according to    claim 1    wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.  
     
     
         8 . The energy transfer dye according to    claim 1    wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichiororhodamine dyes, carboxyrhodamines, N,N,N′,N′-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines and Cy5.  
     
     
         9 . The energy transfer dye according to    claim 1    wherein the acceptor dye is selected from the group consisting of R110, RG6, TAMRA and ROX.  
     
     
         10 . The energy transfer dye according to    claim 1    further comprising a linker attaching the donor dye to the acceptor dye.  
     
     
         11 . The energy transfer dye according to    claim 10    wherein the linker linking the donor dye to the acceptor dye is such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker including a functional group 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.  
     
     
         12 . The energy transfer dye according to    claim 11    wherein the linker functional group is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.  
     
     
         13 . An energy transfer dye comprising: 
 a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure; and    an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.    
     
     
         14 . The energy transfer dye according to    claim 13    wherein the donor dye has an absorption maxima at a wavelength between about 250 and 450 nm.  
     
     
         15 . The energy transfer dye according to    claim 13    wherein the donor dye has an absorption maxima at a wavelength between about 300 and 450nm.  
     
     
         16 . The energy transfer dye according to    claim 13    wherein the donor dye has an absorption maxima at a wavelength between about 350 and 400 nm.  
     
     
         17 . The energy transfer dye according to    claim 13    wherein the acceptor dye has an emission maxima at a wavelength greater than about 550 nm.  
     
     
         18 . The energy transfer dye according to    claim 13    wherein the acceptor dye has an emission maxima at a wavelength between about 500 and 700 nm.  
     
     
         19 . The energy transfer dye according to    claim 13    wherein the acceptor dye has an emission maxima at a wavelength at least about 150 nm greater than the absorption maxima of the donor dye.  
     
     
         20 . The energy transfer dye according to    claim 13    wherein the donor dye is selected from the group consisting of coumarin, pyrene and pyrene sulfonate.  
     
     
         21 . The energy transfer dye according to    claim 13    wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.  
     
     
         22 . The energy transfer dye according to    claim 13    wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′,N′-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines and Cy5.  
     
     
         23 . The energy transfer dye according to    claim 13    wherein the acceptor dye is selected from the group consisting of R110, RG6, TAMRA and ROX.  
     
     
         24 . The energy transfer dye according to    claim 13    further comprising a linker attaching the donor dye to the acceptor dye.  
     
     
         25 . The energy transfer dye according to    claim 24    wherein the linker linking the donor dye to the acceptor dye is such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker including a functional group 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.  
     
     
         26 . The energy transfer dye according to    claim 25    wherein the linker functional group is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.  
     
     
         27 . An energy transfer dye selected from the group consisting of DYE102, DYE104, DYE106, DYE108, DYE118 and DYE120.  
     
     
         28 . A fluorescently labeled reagent comprising: 
 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 donor dye having an absorption maxima at a wavelength between about 250 to 450 nm; and    an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.    
     
     
         29 . The energy transfer dye according to    claim 28    wherein the donor dye has an absorption maxima at a wavelength between about 300 and 450 nm.  
     
     
         30 . The fluorescently labeled reagent according to    claim 28    wherein the donor dye has an absorption maxima at a wavelength between about 350 and 400 nm.  
     
     
         31 . The fluorescently labeled reagent according to    claim 28    wherein the acceptor dye has an emission maxima at a wavelength greater than about 550 nm.  
     
     
         32 . The fluorescently labeled reagent according to    claim 28    wherein the acceptor dye has an emission maxima at a wavelength between about 500 and 700 nm.  
     
     
         33 . The fluorescently labeled reagent according to    claim 28    wherein the acceptor dye has an emission maxima at a wavelength at least about 150 nm greater than the absorption maxima of the donor dye.  
     
     
         34 . The fluorescently labeled reagent according to    claim 28    wherein the acceptor dye is is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.  
     
     
         35 . The fluorescently labeled reagent according to    claim 28    wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichlororhodamine dyes, carboxyrhodamines, N,N,N′ 5 ,N′-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines and Cy5.  
     
     
         36 . The fluorescently labeled reagent according to    claim 28    wherein the acceptor dye is selected from the group consisting of R 110, RG6, TAMRA and ROX.  
     
     
         37 . The fluorescently labeled reagent according to    claim 28    further comprising 
 a linker attaching the donor dye to the acceptor dye.  
 
     
     
         38 . The fluorescently labeled reagent according to    claim 37    wherein the linker linking the donor dye to the acceptor dye is such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker including a functional group 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.  
     
     
         39 . The fluorescently labeled reagent according to    claim 38    wherein the linker functional group is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.  
     
     
         40 . The fluorescently labeled reagent according to    claim 28    wherein the reagent is selected from the group consisting of deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.  
     
     
         41 . The fluorescently labeled reagent according to    claim 40    wherein the deoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.  
     
     
         42 . The fluorescently labeled reagent according to    claim 28    wherein the reagent is selected from the group consisting of dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.  
     
     
         43 . The fluorescently labeled reagent according to    claim 42    wherein the dideoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.  
     
     
         44 . The fluorescently labeled reagent according to    claim 28    wherein the reagent is an oligonucleotide.  
     
     
         45 . The fluorescently labeled reagent according to    claim 44    wherein the oligonucleotide has a 3′ end which is extendable by using a polymerase.  
     
     
         46 . A fluorescently labeled reagent comprising: 
 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 donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure; and    an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.    
     
     
         47 . The fluorescently labeled reagent according to    claim 46    wherein the donor dye has an absorption maxima at a wavelength between about 250 and 450 nm.  
     
     
         48 . The fluorescently labeled reagent according to    claim 46    wherein the donor dye has an absorption maxima at a wavelength between about 300 and 450 nm.  
     
     
         49 . The fluorescently labeled reagent according to    claim 46    wherein the donor dye has an absorption maxima at a wavelength between about 350 and 400 nm.  
     
     
         50 . The fluorescently labeled reagent according to    claim 46    wherein the acceptor dye has an emission maxima at a wavelength greater than about 550 nm.  
     
     
         51 . The fluorescently labeled reagent according to    claim 46    wherein the acceptor dye has an emission maxima at a wavelength between about 500 and 700 nm.  
     
     
         52 . The fluorescently labeled reagent according to    claim 46    wherein the acceptor dye has an emission maxima at a wavelength at least about 150 nm greater than the absorption maxima of the donor dye.  
     
     
         53 . The fluorescently labeled reagent according to    claim 46    wherein the donor dye is selected from the group consisting of coumarin, pyrene and pyrene sulfonate.  
     
     
         54 . The fluorescently labeled reagent according to    claim 46    wherein the acceptor dye is a member of a class of dyes selected from the group consisting of fluorescein, rhodamine, asymmetric benzoxanthene, xanthene, cyanine, phthalocyanine and squaraine dyes.  
     
     
         55 . The fluorescently labeled reagent according to    claim 46    wherein the acceptor dye is selected from the group consisting of 4,7-dichlorofluorescein dyes, asymmetric benzoxanthene dyes, rhodamine, 4,7-dichiororhodamine dyes, carboxyrhodamines, N,N,N′,N′-tetramethyl carboxyrhodamines, carboxy R110, carboxy R6G, carboxy-X-rhodamines and Cy5.  
     
     
         56 . The fluorescently labeled reagent according to    claim 46    wherein the acceptor dye is selected from the group consisting of R110, RG6, TAMRA and ROX.  
     
     
         57 . The fluorescently labeled reagent according to    claim 46    further comprising 
 a linker attaching the donor dye to the acceptor dye.  
 
     
     
         58 . The fluorescently labeled reagent according to    claim 57    wherein the linker linking the donor dye to the acceptor dye is such that the acceptor dye absorbs substantially all of the excitation energy emitted by the donor dye, the linker including a functional group 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.  
     
     
         59 . The fluorescently labeled reagent according to    claim 58    wherein the linker functional group is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.  
     
     
         60 . The fluorescently labeled reagent according to    claim 46    wherein the reagent is selected from the group consisting of deoxynucleoside, deoxynucleoside monophosphate, deoxynucleoside diphosphate and deoxynucleoside triphosphate.  
     
     
         61 . The fluorescently labeled reagent according to    claim 60    wherein the deoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.  
     
     
         62 . The fluorescently labeled reagent according to    claim 46    wherein the reagent is selected from the group consisting of dideoxynucleoside, dideoxynucleoside monophosphate, dideoxynucleoside diphosphate and dideoxynucleoside triphosphate.  
     
     
         63 . The fluorescently labeled reagent according to    claim 62    wherein the dideoxynucleotides are selected from the group consisting of deoxycytosine, deoxyadenosine, deoxyguanosine, and deoxythymidine.  
     
     
         64 . The fluorescently labeled reagent according to    claim 46    wherein the reagent is an oligonucleotide.  
     
     
         65 . The fluorescently labeled reagent according to    claim 64    wherein the oligonucleotide has a 3′ end which is extendable by using a polymerase.  
     
     
         66 . A fluorescently labeled reagent comprising: 
 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 DYE102, DYE104, DYE106, DYE108, DYE118 and DYE120.    
     
     
         67 . A method for sequencing a nucleic acid sequence comprising: 
 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 including 
 a donor dye having an absorption maxima at a wavelength between about 250 to 450 nm; and  
 an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.  
   
     
     
         68 . A method for sequencing a nucleic acid sequence comprising: 
 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 including  
 a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure; and  
 an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.  
   
     
     
         69 . A method for sequencing a nucleic acid sequence comprising: 
 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 including  
 a donor dye having an absorption maxima at a wavelength between about 250 to 450 nm; and  
 an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.  
   
     
     
         70 . A method for sequencing a nucleic acid sequence comprising: 
 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 including  
 a donor dye which is a member of a class of dyes having a coumarin or pyrene ring structure; and  
 an acceptor dye capable of absorbing excitation energy emitted by the donor dye and emitting light in response, the acceptor dye having an emission maxima greater than about 500 nm.