Fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer
First Claim
1. A multichromophore complex 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;
iii) at least one linker group of 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 bonding group capable of forming a covalent bond with a target compound;
wherein said first or second fluorochromes are selected from the group consisting of polymethine cyanine dyes, monomethine rigidized cyanine dyes, fluorescein, pyrene trisulphonate, rhodamine, and bispyrromethine boron difluoride dyes, wherein at least one of said first or second fluorochromes is a cyanine dye, and the combined molecular weight of said first or second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons.
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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
17 Claims
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1. A multichromophore complex 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 of 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 bonding group capable of forming a covalent bond with a target compound;
wherein said first or second fluorochromes are selected from the group consisting of polymethine cyanine dyes, monomethine rigidized cyanine dyes, fluorescein, pyrene trisulphonate, rhodamine, and bispyrromethine boron difluoride dyes, wherein at least one of said first or second fluorochromes is a cyanine dye, and the combined molecular weight of said first or second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification