HOMO-DOUBLY LABELED COMPOSITIONS FOR THE DETECTION OF ENZYME ACTIVITY IN BIOLOGICAL SAMPLES
First Claim
1. A method of delivering a molecule into a cell, said method comprising:
- providing said molecule having attached thereto a first and second fluorophore molecule whereby said first and second fluorophore molecule are the same species of fluorophore and form an H dimer; and
contacting said cell with said molecule whereby said molecule enters said cell.
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Abstract
The present invention provides for novel reagents whose fluorescence changes upon cleavage or a change in conformation of a backbone. The reagents comprise a backbone (e.g. nucleic acid, polypeptide, etc.) joining two fluorophores of the same species whereby the fluorophores form an H-dimer resulting in quenching of the fluorescence of the fluorophores. When the backbone is cleaved or changes conformation, the fluorophores are separated, no longer forming an H-type dimer, and are de-quenched thereby providing a detectable signal. The use of a single fluorophore rather than an “acceptor-donor” fluoresecence resonance energy transfer system offers synthesis and performance advantages.
25 Citations
46 Claims
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1. A method of delivering a molecule into a cell, said method comprising:
- providing said molecule having attached thereto a first and second fluorophore molecule whereby said first and second fluorophore molecule are the same species of fluorophore and form an H dimer; and
contacting said cell with said molecule whereby said molecule enters said cell. - View Dependent Claims (2, 3, 4, 5, 6, 12, 13)
- providing said molecule having attached thereto a first and second fluorophore molecule whereby said first and second fluorophore molecule are the same species of fluorophore and form an H dimer; and
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7-11. -11. (canceled)
- 14. A mammalian cell comprising a biomolecule joining two identical fluorophores whereby said fluorophores form an H-dimer.
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24-28. -28. (canceled)
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29. A method of detecting the activity of a nuclease or the presence of a nucleic acid, said method comprising:
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i) contacting said nuclease or said nucleic acid with a fluorogenic composition comprising a nucleic acid backbone joining two fluorophores of the same species whereby said fluorophores form an H-dimer resulting in quenching of the fluorescence of said fluorophores; and ii) detecting a change in fluorescence or absorbance of said fluorogenic composition where an increase in fluorescence or a change in absorbance indicates that said nuclease cleaves said nucleic acid backbone or that said nucleic acid hybridizes to said backbone. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46)
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Specification