Fret protease assays for clostridial toxins

Abstract

The present invention provides clostridial toxin substrates useful in assaying for the protease activity of any clostridial toxin, including botulinum toxins of all serotypes as well as tetanus toxins. A clostridial toxin substrate of the invention contains a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.

1 . A Clostridial toxin substrate, comprising: 
 a) a donor fluorophore;    b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore; and    c) 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 either of said donor fluorophore, said acceptor, or both said donor fluorophore and said acceptor are genetically encoded; and    wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore;    
     
     
         2 . The substrate of  claim 1 , wherein said donor fluorophore is genetically encoded.  
     
     
         3 . The substrate of  claim 1 , wherein said acceptor is genetically encoded.  
     
     
         4 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are genetically encoded.  
     
     
         5 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 400 residues.  
     
     
         6 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 300 residues.  
     
     
         7 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 200 residues.  
     
     
         8 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 100 residues.  
     
     
         9 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 50 residues.  
     
     
         10 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 40 residues.  
     
     
         11 . The substrate of  claim 1 , which is a peptide or peptidomimetic having at most 20 residues.  
     
     
         12 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most forty residues.  
     
     
         13 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most thirty residues.  
     
     
         14 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most twenty residues.  
     
     
         15 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most fifteen residues.  
     
     
         16 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most ten residues.  
     
     
         17 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most eight residues.  
     
     
         18 . The substrate of  claim 1 , wherein said donor fluorophore and said acceptor are separated by at most six residues.  
     
     
         19 . The substrate of  claim 1 , wherein said substrate can be cleaved with an activity of at least 1 nanomoles/minute/milligram toxin.  
     
     
         20 . The substrate of  claim 1 , wherein said substrate can be cleaved with an activity of at least 20 nanomoles/minute/milligram toxin.  
     
     
         21 . The substrate of  claim 1 , wherein said substrate can be cleaved with an activity of at least 50 nanomoles/minute/milligram toxin.  
     
     
         22 . The substrate of  claim 1 , wherein said substrate can be cleaved with an activity of at least 100 nanomoles/minute/milligram toxin.  
     
     
         23 . The substrate of  claim 1 , wherein said substrate can be cleaved with an activity of at least 150 nanomoles/minute/milligram toxin.  
     
     
         24 . A method of determining Clostridial toxin protease activity, the method comprising the steps of: 
 a) treating a sample, under conditions suitable for Clostridial toxin protease activity, with a Clostridial toxin substrate, said Clostridial toxin substrate according to any one of claims  1 - 23 ;    b) exciting said donor fluorophore; and    c) determining resonance energy transfer of said treated substrate relative to a control substrate,    wherein a difference in resonance energy transfer of said treated substrate as compared to said control substrate is indicative of Clostridial toxin protease activity.    
     
     
         25 . The method of  claim 24 , wherein said sample is a crude cell lysate.  
     
     
         26 . The method of  claim 24 , wherein said sample is isolated Clostridial toxin or isolated Clostridial toxin light chain.  
     
     
         27 . The method of  claim 24 , wherein said sample is a formulated Clostridial toxin product.  
     
     
         28 . The method of  claim 24 , wherein said acceptor is a fluorophore and step (c) comprises detecting donor fluorescence intensity of said treated substrate, wherein an increase in substrate cleavage results in an increase in donor fluorescence intensity of said treated substrate as compared to said control substrate, said increased donor fluorescence intensity being indicative of Clostridial toxin protease activity.  
     
     
         29 . The method of  claim 24 , wherein said acceptor is a fluorophore and step (c) comprises detecting acceptor fluorescence intensity of said treated substrate, wherein an increase in substrate cleavage results in a decrease in acceptor fluorescence intensity of said treated substrate as compared to said control substrate, said decreased acceptor fluorescence intensity being indicative of Clostridial toxin protease activity.  
     
     
         30 . The method of  claim 24 , wherein said acceptor is a fluorophore and step (c) comprises detecting an acceptor emission maximum and a donor fluorophore emission maximum, wherein an increase in substrate cleavage results in a shift in emission maxima from near said acceptor emission maximum to near said donor fluorophore emission maximum, said shift in emission maxima being indicative of Clostridial toxin protease activity.  
     
     
         31 . The method of  claim 24 , wherein said acceptor is a fluorophore and step (c) comprises detecting the ratio of fluorescence amplitudes near an acceptor emission maximum over the fluorescence amplitudes near a donorfluorophore emission maximum, wherein an increase in substrate cleavage results in a decreased ratio of said treated substrate as compared to said control substrate, said decreased ratio being indicative of Clostridial toxin protease activity.  
     
     
         32 . The method of  claim 24 , wherein said acceptor is a fluorophore and step (c) comprises detecting the ratio of fluorescence amplitudes near an donor emission maximum over the fluorescence amplitudes near a acceptor fluorophore emission maximum, wherein an increase in substrate cleavage results in an increased ratio in said treated substrate as compared to the control substrate, said increased ratio being indicative of Clostridial toxin protease activity.  
     
     
         33 . The method of  claim 24 , wherein said acceptor is a fluorophore and step (c) comprises detecting the excited state lifetime of the donor fluorophore of said treated substrate, wherein an increase in substrate cleavage results in an increase in donor fluorophore excited state lifetime of said treated substrate as compared to said control substrate, said increased excited state lifetime being indicative of Clostridial toxin protease activity.  
     
     
         34 . The method of  claim 24 , wherein said acceptor is a quencher and step (c) comprises detecting donor fluorescence intensity of said contacted cell, wherein an increase in substrate cleavage results in an increase in donor fluorescence intensity of said treated substrate as compared to said control substrate, said increased donor fluorescence intensity being indicative of Clostridial toxin protease activity.  
     
     
         35 . The method of  claim 24 , further comprising repeating step (c) at one or more later time intervals.  
     
     
         36 . The method of  claim 24 , wherein at least 90% of said Clostridial toxin substrate is cleaved.  
     
     
         37 . The method of  claim 24 , wherein at most 25% of said Clostridial toxin substrate is cleaved.  
     
     
         38 . The method of  claim 24 , wherein at most 15% of said Clostridial toxin substrate is cleaved.  
     
     
         39 . The method of  claim 24 , wherein at most 5% of said Clostridial toxin substrate is cleaved.  
     
     
         40 . The method of  claim 24 , wherein the conditions suitable for Clostridial toxin protease activity are selected such that the assay is linear.