Fluorescent labeling complexes with large Stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer
First Claim
1. A set of fluorescent labeling complexes each of said complexes comprising:
- i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.
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Abstract
The present invention provides low molecular weight fluorescent labeling complexes with large wavelength shifts between absorption of one dye in the complex and emission from another dye in the complex. These complexes can be used, for example, for multiparameter fluorescence cell analysis using a single excitation wavelength. The low molecular weight of the complex permits materials labeled with the complex to penetrate cell structures for use as probes. The labeling complexes are synthesized by covalently attaching through linkers at least one cyanine fluorochrome to another low molecular weight fluorochrome to form energy donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300 nm. The fluorescent labeling complexes preferably contain reactive groups for the labeling of functional groups on target compounds, such as derivatized oxy and deoxy polynucleic acids, antibodies, enzymes, proteins and other materials. The complexes may also contain functional groups permitting covalent reaction with materials containing reactive groups.
60 Citations
29 Claims
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1. A set of fluorescent labeling complexes each of said complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 16, 18, 19, 22)
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9. A set of fluorescent labeling complexes each of said complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
iii) at least one linker group chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochomes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound;
wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye and wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons. - View Dependent Claims (17, 20, 21, 23)
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10. A set of reagents each reagent comprising:
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A) a fluorescent water soluble labeling complex comprised of;
i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker group to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome;
ii) at least one target bonding group capable of forming a covalent bond with a carrier material; and
,iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one target bonding group;
wherein at least one of said first or second fluorochromes is a cyanine dye and the linker group is chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CO—
NH groups as amide linkages;B) a carrier material having a group that reacts with said target bonding group of said complex and is covalently bound thereto. - View Dependent Claims (11, 12)
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13. A method of labeling a carrier material with one of a set of fluorescent labeling complexes, comprising incubating an aqueous sample containing a carrier material with a low molecular weight, water soluble fluorescent labeling complex comprised of:
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i) a first fluorochrome having first absorption and emission spectra covalently linked through a linker group to a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and the absorption spectrum of said second fluorochrome overlapping the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome, wherein at least one of said first or second fluorochromes in said complex is a cyanine dye;
ii) a target bonding group attached to said complex capable of forming a covalent bond with a complementary group of said carrier material, and iii) water solubilizing constituents attached to said complex for conferring a polar characteristic to said complex, said water solubilizing constituents being unreactive with said bonding group;
for a period of time sufficient for covalently binding said bonding group of said complex to said complementary group of said carrier material. - View Dependent Claims (24)
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14. Use of a set of fluorescent labeling complexes for analysis or detection comprising incubating a fluorescent labeling complex of said set with at least one target material, each of said fluorescent labeling complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least one of said first and second fluorochromes in each of said complexes is a cyanine dye, and wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths; and
measuring and comparing the differences in fluorescent emission between said fluorescent labeling complexes. - View Dependent Claims (25)
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15. Use of a set of fluorescent labeling complexes for analysis and detection comprising incubating a fluorescent labeling complex of said set with at least one target material, each of said fluorescent labeling complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
iii) at least one linker group chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye; and
measuring and comparing the differences in fluorescent emission between said fluorescent labeling complexes. - View Dependent Claims (26)
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27. A set of fluorescent labeling complexes each of said complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein said fluorochromes and said linker in each of said complexes, the relative orientation of the transition moments of the fluorochromes during the excited state lifetime of the first fluorochrome, and the proximity of the fluorochromes, are selected such that there is sufficient energy transfer; wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.
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28. A set of fluorescent labeling complexes each of said complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes, wherein said linker group optimally orients the transition moments of said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.
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29. A set of fluorescent labeling complexes each of said complexes comprising:
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i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes, wherein said linker group is rigid;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.
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Specification