Homo-doubly labeled compositions for the detection of enzyme activity in biological samples
First Claim
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1. A fluorogenic composition comprising a polypeptide backbone or 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.
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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.
51 Citations
102 Claims
- 1. A fluorogenic composition comprising a polypeptide backbone or 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.
- 32. A mammalian cell comprising a fluorogenic composition comprising a polypeptide backbone or a nucleic acid backbone joining two identical fluorophores whereby said fluorophores form an H-dimer resulting in the quenching of the fluorescence of said fluorophores.
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48. A method of detecting the activity of a protease, said method comprising:
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i) contacting said protease with a fluorogenic composition comprising a polypeptide 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 protease cleaves said polypeptide backbone. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65)
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66. 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 (67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83)
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84. A method of detecting the interaction of a first and a second molecule, said method comprising:
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i) providing a first molecule having a first fluorophore attached thereto;
ii) providing a second molecule having a second fluorophore attached thereto wherein said first fluorophore and said second fluorophore are the same species of fluorophore and, when juxtaposed, form an H-dimer thereby quenching fluorescence produced by the fluorophores; and
iii) detecting a change in fluorescence or absorbance produced by said fluorophores where a decrease in fluorescence or a change in absorbance indicates that the first molecule and the second molecule are interacting. - View Dependent Claims (85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102)
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89. A method of detecting a change in conformation or cleavage of a macromolecule, said method comprising:
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i) providing a macromolecule having attached thereto 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 fluorophores wherein a change in fluorescence or absorbance indicates a change in conformation or cleavage of said macromolecule.
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Specification
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Current AssigneeOncolmmunin Incorporated
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Original AssigneeOncolmmunin Incorporated
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InventorsKomoriya, Akira, Packard, Beverly
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Granted Patent
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Time in Patent OfficeDays
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Field of Search
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US Class Current435/6
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CPC Class CodesC07K 7/06 having 5 to 11 amino acidsC07K 7/08 having 12 to 20 amino acids...C12Q 1/37 involving peptidase or prot...C12Q 1/44 involving esteraseG01N 2333/918 Carboxylic ester hydrolases...G01N 2333/96425 from mammalsG01N 2333/96469 Interleukin 1-beta converta...G01N 33/533 with fluorescent labelG01N 33/542 with steric inhibition or s...
