Cell-based fluorescence resonance energy transfer (fret) assays for clostridial toxins

Abstract

The present invention provides a method of determining clostridial toxin activity by (a) contacting with a sample a cell containing a clostridial toxin substrate that includes a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence containing a cleavage site that intervenes between the donor fluorophore and the acceptor, where resonance energy transfer is exhibited between the donor fluorophore and the acceptor under the appropriate conditions; (b) exciting the donor fluorophore; and (c) determining resonance energy transfer of the contacted cell relative to a control cell, where a difference in resonance energy transfer of the contacted cell as compared to the control cell is indicative of clostridial toxin activity.

1 . A cell comprising 
 (a) at least one receptor that binds a clostridial toxin, and    (b) a clostridial toxin substrate, said clostridial toxin substrate comprising: 
 (i) a donor fluorophore;  
 (ii) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore; and  
 (iii) a clostridial toxin recognition sequence comprising a clostridial toxin recognition sequence comprising a clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence, said clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence intervening between said donor fluorophore and said acceptor,  
   wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.    
     
     
         2 . The cell of  claim 1 , wherein said cell is a neuron.  
     
     
         3 . The cell of  claim 2 , wherein said neuron is a peripheral neuron or a central nervous system neuron.  
     
     
         4 . The cell of  claim 2 , wherein said neuron is a selected from the group consisting of a primary cell, a cultured cell, an established cell, a normal cell, a transformed cell, a tumor cell, an infected cell and a transfected cell.  
     
     
         5 . The cell of  claim 4 , wherein said established line is a neuronal cell line selected from the group consisting of a neuroblastoma cell line, a hybrid neuronal cell line, a motor neuron cell line, a spinal cord cell line, a cerebral cortex cell line, a dorsal root ganglia cell line and a hippocampal cell line.  
     
     
         6 . The cell of  claim 1 , wherein said cell is a non-neuronal cell.  
     
     
         7 . The cell of  claim 6 , wherein said non-neuronal cell is selected from the group consisting of a glandular cell, an anterior pituitary cell, an adrenal cell, a pancreatic cell, an epithelial cell, a muscle cell, a fibroblast, a neutrophil, an eosinophil, a mast cell, a stomach cell, a hepatocyte, a kidney cell and an ovarian cell.  
     
     
         8 . The cell of  claim 6 , wherein said non-neuronal cell is a selected from the group consisting of a primary cell, a cultured cell, an established cell, a normal cell, a transformed cell, a tumor cell, an infected cell and a transfected cell.  
     
     
         9 . The cell of  claim 8 , wherein said established line is a non-neuronal cell line selected from the group consisting of a chromaffin cell line, an enterochromaffin cell line, a pancreatic islet β cell line, a pancreatic acinar cell line, an insulinoma HIT cell line, an INS-1 cell line, a steroid-producing ovarian cell line, an inner medullary collecting duct (IMCD) cell line, a platelet cell line, a neutrophil cell line, an eosinophil cell line, a mast cell line and a glucose transporter translocation cell line.  
     
     
         10 . The cell of  claim 1 , wherein said receptor is selected from the group consisting of a high affinity receptor, a low affinity receptor, an endogenous receptor and an exogenous receptor.  
     
     
         11 . The cell of  claim 1 , wherein said donor fluorophore is selected from the group consisting of blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, yellow fluorescent protein and red fluorescent protein.  
     
     
         12 . The cell of  claim 1 , wherein said acceptor is a fluorophore selected from the group consisting of blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, yellow fluorescent protein and red fluorescent protein.  
     
     
         13 . The cell of  claim 1 , wherein said acceptor is a non-fluorescent molecule.  
     
     
         14 . The cell of  claim 1 , wherein said clostridial toxin substrate is a botulinum toxin substrate, wherein said clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a botulinum toxin recognition sequence comprising a botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         15 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/A substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/A recoginition sequence comprising a BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         16 . The cell of  claim 15 , wherein said BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site comprises at least six consecutive residues of SNAP-25 or a peptidomimetic thereof, said six consecutive residues comprising Gln-Arg, or a peptidomimetic thereof.  
     
     
         17 . The cell of  claim 16 , wherein said BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 41.  
     
     
         18 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/B substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/B recognition sequence comprising a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         19 . The cell of  claim 18 , wherein said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of VAMP, or a peptidomimetic thereof, said six consecutive residues comprising Gln-Phe, or a peptidomimetic thereof.  
     
     
         20 . The cell of  claim 19 , wherein said BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site region sequence comprises SEQ ID NO: 42.  
     
     
         21 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/C1 substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/C1 recognition sequence comprising a BoNT/C1 P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         22 . The cell of  claim 21 , wherein said BoNT/C1 P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of syntaxin, or a peptidomimetic thereof, said six consecutive residues comprising Lys-Ala, or a peptidomimetic thereof.  
     
     
         23 . The cell of  claim 21 , wherein said BoNT/C1 P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of SNAP-25, or a peptidomimetic thereof, said six consecutive residues comprising Arg-Ala, or a peptidomimetic thereof.  
     
     
         24 . The cell of  claim 23 , wherein said BoNT/C1 P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 43.  
     
     
         25 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/D substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/D recognition sequence comprising a BoNT/D P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         26 . The cell of  claim 25 , wherein said BoNT/D P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of VAMP, or a peptidomimetic thereof, said six consecutive residues comprising Lys-Leu, or a peptidomimetic thereof.  
     
     
         27 . The cell of  claim 26 , wherein said BoNT/D P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 44.  
     
     
         28 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/E substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/E recognition sequence comprising a BoNT/E P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         29 . The cell of  claim 28 , wherein said BoNT/E P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of SNAP 25, or a peptidomimetic thereof, said six consecutive residues comprising Arg-Ile, or a peptidomimetic thereof.  
     
     
         30 . The cell of  claim 29 , wherein said BoNT/E P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 45.  
     
     
         31 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/F substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/F recognition sequence comprising a BoNT/F P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         32 . The cell of  claim 31 , wherein said BoNT/F P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of VAMP, or a peptidomimetic thereof, said six consecutive residues comprising Gln-Lys, or a peptidomimetic thereof.  
     
     
         33 . The cell of  claim 32 , wherein said BoNT/F P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 46.  
     
     
         34 . The cell of  claim 14 , wherein said botulinum toxin substrate is a BoNT/G substrate, wherein said botulinum toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a BoNT/G recognition sequence comprising a BoNT/G P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         35 . The cell of  claim 34 , wherein said BoNT/G P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of VAMP, or a peptidomimetic thereof, said six consecutive residues comprising Ala-Ala, or a peptidomimetic thereof.  
     
     
         36 . The cell of  claim 35 , wherein said BoNT/G P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 47.  
     
     
         37 . The cell of  claim 1 , wherein said clostridial toxin substrate is a TeNT toxin substrate, wherein said clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence is a TeNT recognition sequence comprising a TeNT P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence.  
     
     
         38 . The cell of  claim 37 , wherein said TeNT P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises at least six consecutive residues of VAMP, or a peptidomimetic thereof, said six consecutive residues comprising Gln-Phe, or a peptidomimetic thereof.  
     
     
         39 . The cell of  claim 38 , wherein said TeNT P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ cleavage site sequence comprises SEQ ID NO: 48.  
     
     
         40 . The cell of  claim 1 , wherein said clostridial toxin substrate comprises only one clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         41 . The cell of  claim 1 , wherein said clostridial toxin substrate comprises multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 -P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences from the same clostridial toxin.  
     
     
         42 . The cell of  claim 1 , wherein said clostridial toxin substrate comprises multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences from different clostridial toxins.  
     
     
         43 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         44 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence and a BoNT/C1 P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         45 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence, a BoNT/C1 P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence and a BoNT/E P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         46 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         47 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence and a BoNT/G P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         48 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/D P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         49 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/D P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence and a BoNT/F P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         50 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence, a BoNT/D P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence, a BoNT/F P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence and a BoNT/G P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         51 . The cell of  claim 42 , wherein said multiple clostridial toxin P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequences comprise a BoNT/A P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence and a BoNT/B P 5 -P 4 -P 3 -P 2 -P 1 -P 1 ′-P 2 ′-P 3 ′-P 4 ′-P 5 ′ recognition sequence.  
     
     
         52 . The cell of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most 70 residues.  
     
     
         53 . The cell of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most 100 residues.  
     
     
         54 . The cell of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most 150 residues.  
     
     
         55 . The cell of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most 200 residues.  
     
     
         56 . The cell of  claim 1 , wherein said donor fluorophore and said acceptor are separated by the full length of a naturally-occurring clostridial toxin target protein.  
     
     
         57 . The cell of  claim 1 , wherein said clostridial toxin substrate of any one of claims  1 , 11-56 is transiently transfected into said cell.  
     
     
         58 . The cell of  claim 1 , wherein said clostridial toxin substrate of any one of claims  1 , 11-56 is encoded by a nucleic acid molecule.  
     
     
         59 . The cell of  claim 58 , wherein said nucleic acid molecule is linked to a regulatory element selected from the group consisting of a constitutive regulatory element and an inducible regulatory element.  
     
     
         60 . The cell of either  claim 58  or  59 , wherein said nucleic acid molecule is transiently transfected into said cell.  
     
     
         61 . The cell of either  claim 58  or  59 , wherein said nucleic acid molecule is stably transfected into said cell.