Energy transfer labels with mechanically linked fluorophores
First Claim
1. An energy transfer label comprising at least one donor fluorophore, at least one acceptor fluorophore, and at least one support member, wherein steric interactions between two or more of said donor fluorophore, said acceptor fluorophore, and said support member induce non-covalent association between said donor fluorophore, said acceptor fluorophore, and said support member, thereby forming a macromolecular structure which mechanically links said donor fluorophore and said acceptor fluorophore.
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Accused Products
Abstract
Mechanically linked energy transfer labels comprising at least one donor fluorophore, at least one acceptor fluorophore, and at least one support member, wherein steric interactions between the donor fluorophore(s), the acceptor fluorophore(s), and/or the support member(s) induce non-covalent association between the fluorophores and the support member(s), thereby forming a three-dimensional macromolecular structure which mechanically links the donor fluorophore(s) and the acceptor fluorophore(s). Fluorescence resonance energy transfer (FRET) occurs from donor fluorophore to acceptor fluorophore through space. No direct connectivity with covalent bonds exists between the fluorophores. Instead, mechanical barriers hold the donor/acceptor fluorophores in place during the FRET process.
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Citations
52 Claims
- 1. An energy transfer label comprising at least one donor fluorophore, at least one acceptor fluorophore, and at least one support member, wherein steric interactions between two or more of said donor fluorophore, said acceptor fluorophore, and said support member induce non-covalent association between said donor fluorophore, said acceptor fluorophore, and said support member, thereby forming a macromolecular structure which mechanically links said donor fluorophore and said acceptor fluorophore.
- 42. A method for labeling a biomolecule comprising contacting said biomolecule with an energy transfer label, under conditions suitable to form a covalent bond between said biomolecule and said energy transfer label, thereby forming a labeled biomolecule, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore.
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45. A method for detecting a biomolecule comprising
contacting said biomolecule with an energy transfer label under conditions suitable to form a covalent bond between said biomolecule and said energy transfer label, thereby forming a labeled biomolecule, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore, irradiating said labeled biomolecule at a first wavelength, and detecting energy emission at a second wavelength.
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46. A method for identifying nucleic acids in a multi-nucleic acid mixture comprising
contacting said nucleic acids with a plurality of energy transfer labels under conditions suitable to form a covalent bond between said nucleic acids and said energy transfer labels, thereby forming labeled nucleic acids, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore, and wherein said energy transfer labels comprise donor fluorophores which absorb radiation at a first wavelength and acceptor fluorophores which emit radiation at wavelengths other than said first wavelength, irradiating said labeled nucleic acids at said first wavelength, and detecting energy emission at said wavelengths other than said first wavelength.
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47. A method for sequencing a polynucleotide comprising
forming a mixture of extended labeled primers by hybridizing a polynucleotide with an oligonucleotide primer labeled with an energy transfer label in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate, and a DNA polymerase, wherein the DNA polymerase extends the primer with the deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore, separating said mixture of extended labeled primers, determining the sequence of the polynucleotide by irradiating said mixture of extended labeled primers.
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48. A method for sequencing a polynucleotide comprising
forming a mixture of extended primers by hybridizing a polynucleotide with an oligonucleotide primer in the presence of deoxynucleoside triphosphates, at least one dideoxynucleoside triphosphate labeled with an energy transfer label, and a DNA polymerase, wherein the DNA polymerase extends the primer with the deoxynucleoside triphosphates until a labeled dideoxynucleoside triphosphate is incorporated which terminates extension of the primer, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore, separating the mixture of extended primers, and determining the sequence of the polynucleotide by detecting the labeled dideoxynucleoside triphosphate attached to the extended primers.
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49. A method for sequencing a polynucleotide comprising
forming a mixture of extended primers by hybridizing a polynucleotide with an oligonucleotide primer in the presence of deoxynucleoside triphosphates labeled with an energy transfer label, at least one dideoxynucleoside triphosphate, and a DNA polymerase, wherein the DNA polymerase extends the primer with the labeled deoxynucleoside triphosphates until a dideoxynucleoside triphosphate is incorporated which terminates extension of the primer, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore, separating the mixture of extended primers, and determining the sequence of the polynucleotide by detecting the labeled deoxynucleoside triphosphates attached to the extended primers.
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50. A method for increasing the intensity of a fluorescence resonance energy transfer signal comprising contacting an analyte with an energy transfer label under conditions suitable to form a covalent bond between said analyte and said energy transfer label, wherein said energy transfer label comprises at least one donor fluorophore covalently attached to a first support member and at least one acceptor fluorophore covalently attached to a second support member, wherein steric interactions between said support members mechanically link said donor fluorophore and said acceptor fluorophore, thereby forming a labeled analyte, irradiating said analyte at a first wavelength, and detecting energy emission at wavelengths other than said first wavelength.
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51. An energy transfer label comprising a plurality of donor fluorophores, at least one acceptor fluorophore, and at least one support member, wherein steric interactions between two or more of said donor fluorophore, said acceptor fluorophore, and said support member induce non-covalent association between said donor fluorophore, said acceptor fluorophore, and said support member, thereby forming a macromolecular structure which mechanically links said donor fluorophore and said acceptor fluorophore.
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52. An energy transfer label comprising at least one donor fluorophore, a plurality of acceptor fluorophores, and at least one support member, wherein steric interactions between two or more of said donor fluorophore, said acceptor fluorophore, and said support member induce non-covalent association between said donor fluorophore, said acceptor fluorophore, and said support member, thereby forming a macromolecular structure which mechanically links said donor fluorophore and said acceptor fluorophore.
Specification