US 5,945,526 A · Energy transfer dyes with enhanced fluorescence

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 R.sub.21 Z.sub.1 C(O)R.sub.22 R.sub.28 where R.sub.21 is a C.sub.1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z.sub.1 is either NH, sulfur or oxygen, R.sub.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.sub.28 includes a functional group which attaches the linker to the acceptor dye.

Claims109

Dependent claims

An energy transfer dye having the structure ##STR120## 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;
Z.sub.1 is selected from the group consisting of NH, sulfur and oxygen;
R.sub.21 is a C.sub.1-5 alkyl attached to the donor dye;
R.sub.22 includes 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 which is attached to the carbonyl carbon;
and
R.sub.28 includes a functional group which attaches the linker to the acceptor dye.

3 more elements

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 dye having the structure ##STR129## where Y.sub.1 and Y.sub.2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;
Z.sub.1 is selected from the group consisting of NH, sulfur and oxygen;
R.sub.11 -R.sub.17 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;
R.sub.21 is a C.sub.1-5 alkyl;
R.sub.22 includes 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 which is attached to the carbonyl carbon;
R.sub.28 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.

7 more elements

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 dye having the structure ##STR132## wherein:
Y.sub.1, Y.sub.1 ', Y.sub.2 ' and Y.sub.2 ' are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine,
R.sub.11 -R.sub.16 and R.sub.11 '-R.sub.16 ' 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
X.sub.1 -X.sub.5 and X.sub.1 '-X.sub.5 ' 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;
Y.sub.1, Y.sub.2, R.sub.11 -R.sub.16, and X.sub.1 -X.sub.5 are selected to correspond to a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;
Y.sub.1 ', Y.sub.2 ', R.sub.11 '-R.sub.16 ', and X.sub.1 '-X.sub.5 ' 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 X.sub.3 and X.sub.4 and one of X.sub.3 ' and X.sub.4 ' are taken together to form a linker one of X.sub.3 and X.sub.4 and one of X.sub.3 ' and X.sub.4 ' are taken together to form a linker having the general formula R.sub.25 Z.sub.3 C(O)R.sub.37 where R.sub.25 is a C.sub.1-4 alkyl attached to the donor dye at the X.sub.3 or X.sub.4 substituent, Z.sub.3 is either NH, O or S, C(O) is a carbonyl group and R.sub.37 is a substituent attached to the acceptor dye at the X.sub.3 ' or X.sub.1 ' substituent.

7 more elements

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 having the structure ##STR134## where Y.sub.1 and Y.sub.2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;
Z.sub.1 is selected from the group consisting of NH, sulfur and oxygen;
R.sub.11 -R.sub.17 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;
R.sub.2 1 is a C.sub.1-5 alkyl;
R.sub.22 includes 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 which is attached to the carbonyl carbon;
R.sub.28 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.

10 more elements

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 dye having the structure ##STR136## where Y.sub.1 and Y.sub.2 are each independently selected from the group consisting of hydroxyl, oxygen, iminium and amine;
Z.sub.1 is selected from the group consisting of NH, sulfur and oxygen;
R.sub.11 -R.sub.17 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;
R.sub.21 is a C.sub.1-5 alkyl;
R.sub.22 includes 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 which is attached to the carbonyl carbon;
R.sub.28 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.

11 more elements

An energy transfer dye comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;
an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;
and
a linker linking the donor dye to the acceptor dye 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.

1 more element

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 capable of absorbing light at a first wavelength and emitting excitation energy in response;
an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;
and
a linker linking the donor dye to the acceptor dye 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.

5 more elements

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 capable of absorbing light at a first wavelength and emitting excitation energy in response,
an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, and
a linker linking the donor dye to the acceptor dye 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.

7 more elements

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 capable of absorbing light at a first wavelength and emitting excitation energy in response,
an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, and
a linker linking the donor dye to the acceptor dye 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.

7 more elements

Dependencies are inferred from the claim text.

Energy transfer dyes with enhanced fluorescence

Life of this patent

Energy transfer dyes with enhanced fluorescence

Life of this patent

Abstract

Claims109