Homo-doubly labeled compositions for the detection of enzyme activity in biological samples

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.

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. 
     
     
         2 . The method of  claim 1 , wherein said molecule is a molecule selected from the group consisting of a polypeptide, an oligonucleotide, an oligosaccharide, and a lipid. 
     
     
         3 . The method of  claim 1 , wherein said molecule further has attached thereto a hydrophobic group. 
     
     
         4 . The method of  claim 3 , wherein said hydrophobic group is selected from the group consisting of Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA). 
     
     
         5 . The method of  claim 1 , wherein the first fluorophore and the second fluorophore have an excitation wavelength between about 310 nm and about 750 nm. 
     
     
         6 . The method of  claim 1 , wherein the first fluorophore and the second fluorophore are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes. 
     
     
         7 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said molecule is a nucleic acid that ranges in length from about 10 to about 100 nucleotides. 
     
     
         13 . The method of  claim 1 , wherein said molecule is a nucleic acid that ranges in length from about 15 to about 50 nucleotides. 
     
     
         14 . A mammalian cell comprising a biomolecule joining two identical fluorophores whereby said fluorophores form an H-dimer. 
     
     
         15 . The cell of  claim 14 , wherein said biomolecule is a nucleic acid. 
     
     
         16 . The cell of  claim 14 , wherein said biomolecule bears a hydrophobic group. 
     
     
         17 . The cell of  claim 16 , wherein said hydrophobic group is selected from the group consisting of: Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA). 
     
     
         18 . The cell of  claim 17 , wherein said hydrophobic group is Fmoc. 
     
     
         19 . The cell of  claim 14 , wherein said hydrophobic group is Fa. 
     
     
         20 . The cell of  claim 14 , wherein said hydrophobic group is attached to the amino terminus of the molecule. 
     
     
         21 . The cell of  claim 14 , wherein said fluorophores are linked to the biomolecule by linkers. 
     
     
         22 . The cell of  claim 14 , wherein said fluorophores have an excitation wavelength between about 310 nm and about 750 nm. 
     
     
         23 . The cell of  claim 14 , wherein said fluorophores are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes. 
     
     
         24 - 28 . (canceled) 
     
     
         29 . A method of detecting the activity of a nuclease or the presence of a nucleic acid, said method comprising:
 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.   
     
     
         30 . The method of  claim 29 , wherein said nucleic acid backbone comprises a restriction site. 
     
     
         31 . The method of  claim 29 , wherein said nucleic acid backbone is self-complementary and forms a hairpin. 
     
     
         32 . The method of  claim 29 , wherein said nucleic acid backbone ranges in length from about 10 to about 100 nucleotides. 
     
     
         33 . The method of  claim 29 , wherein said nucleic acid backbone ranges in length from about 15 to about 50 nucleotides. 
     
     
         34 . The method of  claim 29 , wherein said composition is attached to a solid support. 
     
     
         35 . The method of  claim 29 , wherein said composition is inside a mammalian cell. 
     
     
         36 . The method of  claim 29 , wherein said composition is in solution. 
     
     
         37 . The method of  claim 29 , wherein said composition bears a hydrophobic group. 
     
     
         38 . The method of  claim 37 , wherein said hydrophobic group is selected from the group consisting of: Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, and 9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4′-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys), 1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA). 
     
     
         39 . The method of  claim 29 , wherein said fluorophores are linked to the nucleic acid backbone or to the polypeptide backbone by linkers. 
     
     
         40 . The method of  claim 29 , wherein said fluorophores have an excitation wavelength between about 310 nm and about 750 nm. 
     
     
         41 . The method of  claim 29 , wherein said fluorophores are selected from the group consisting of carboxytetramethylrhodamine, carboxyrhodamine-X, carboxyrhodamine 110, diethylaminocoumarin, and carbocyanine dyes. 
     
     
         42 . The method of  claim 29 , wherein said contacting is in a histological section. 
     
     
         43 . The method of  claim 29 , wherein said contacting is in a cell culture. 
     
     
         44 . The method of  claim 29 , wherein said contacting is contacting a seeded or cultured adherent cell. 
     
     
         45 . The method of  claim 29 , wherein said contacting is in a cell suspension derived from a biological sample selected from the group consisting of a tissue, blood, urine, saliva, lymph, biopsy. 
     
     
         46 . The method of  claim 29 , wherein said detecting is by a method selected from the group consisting of fluorescence microscopy, confocal microscopy, fluorescence microplate reader, flow cytometry, fluorometry, and absorption spectroscopy.