Enzyme assay with duplicate fluorophores

Abstract

Compositions and methods are disclosed that provide a rapid, sensitive, and accurate cell-based assay for enzyme activity, particularly for enzyme activities associated with botulinum toxins. A cell is provided that expresses a construct that includes an anchor region, a cleavage site, and a reporting region having two or more identical reporter peptides. Enzymatic activity at the cleavage site releases the reporter region into the cytosol, where multiple degradation events occur. The observed change in the signal is proportional to the enzymatic activity.

What is claimed is: 
     
         1 . A reporting construct for characterizing an enzyme activity comprising:
 a membrane anchoring domain comprising a first peptide that forms a complex with a membrane of a cell;   a reporter domain comprising first occurrence of a second peptide that produces a first signal at a first wavelength and a second occurrence of the second peptide that produces a second signal at the first wavelength, wherein the reporter domain produces an aggregate signal that is a summation of the first signal and the second signal;   a cleavage site comprising a third peptide, the cleavage site interposed between the membrane anchoring domain and at least two of the two or more occurrences of the reporter domain,   wherein the third peptide is selected to undergo a cleavage event upon exposure to the enzyme activity,   wherein the aggregate signal is observable in the absence of either one of the first signal or the second signal.   
     
     
         2 . The reporting construct of  claim 1 , wherein the first occurrence of the second peptide is susceptible to a first degradative event in the cell cytosol and the second occurrence of the second peptide is susceptible to a second degradative event in the cell cytosol following a cleavage event, wherein the first degradative event results in the loss of the production of the first signal and the second degradative event results in the loss of the production of the second signal. 
     
     
         3 . The reporting construct of  claim 1 , wherein the second peptide is a fluorescent protein. 
     
     
         4 . The reporting construct of  claim 1 , further comprising a linker peptide interposed between the first occurrence of the second peptide and the second occurrence of the second peptide. 
     
     
         5 . The reporting construct of  claim 1 , wherein the second peptide has at least 80% sequence identity to SEQ ID NO. 10. 
     
     
         6 . The reporting construct of  claim 1 , wherein the third peptide is a SNARE protein or a fragment thereof. 
     
     
         7 . The reporting construct of  claim 1  further comprising an auxiliary reporting domain that provides a signal at a second wavelength, wherein the second wavelength is distinguishable from the first wavelength. 
     
     
         8 . The reporting construct of  claim 1 , wherein the reporting construct shows reduced selectivity bias between the first enzyme activity and a second enzyme activity relative to an analogous reporting construct that includes a single occurrence of the second peptide, wherein the third peptide is selected to undergo a cleavage event upon exposure to either of the first enzyme activity or the second enzyme activity. 
     
     
         9 . The reporting construct of  claim 9 , wherein the first enzyme activity is associated with BoNT/A and the second enzyme activity is associated with BoNT/E. 
     
     
         10 . The reporting construct of  claim 1 , wherein the reporting construct does not exhibit useful levels of FRET. 
     
     
         11 . A method of characterizing a first enzyme activity comprising:
 providing a cell that expresses a reporting construct comprising a membrane anchoring domain comprising a first peptide that forms a complex with a membrane of a cell, a reporter domain comprising a first occurrence of a second peptide that produces a first signal at a first wavelength and a second occurrence of the second peptide that produces a second signal at the first wavelength wherein the reporter domain produces an aggregate signal that is a summation of the first signal and the second signal, and a cleavage site comprising a third peptide, the cleavage site interposed between the membrane anchoring domain and at least two of the two or more occurrences of the reporter domain and wherein the third peptide is selected to undergo a cleavage event upon exposure to the first enzyme activity;   contacting the cell with a sample suspected of including the first enzyme activity; and   observing a decrease in the detectable signal when the sample includes the first enzyme activity,   wherein the aggregate signal is observable in the absence of either one of the first signal or the second signal.   
     
     
         12 . The method of  claim 11 , wherein the second peptide is a fluorescent protein. 
     
     
         13 . The method of  claim 11 , wherein the method shows reduced selectivity between the first enzyme activity and a second enzyme activity relative to an assay utilizing an analogous reporting construct that includes a single occurrence of the second peptide, wherein the third peptide is selected to undergo a cleavage event upon exposure to either of the first enzyme activity or the second enzyme activity. 
     
     
         14 . The method of  claim 13 , wherein the first enzyme activity is associated with BoNT/A and the second enzyme activity is associated with BoNT/E. 
     
     
         15 . The method of  claim 11 , wherein the second peptide has at least 80% sequence identity to SEQ ID NO.  10 . 
     
     
         16 . The method of  claim 11 , wherein the third peptide is a SNARE protein or a fragment thereof. 
     
     
         17 . The method of  claim 11  wherein the reporting construct further comprises a reference reporter that produces a reference signal, wherein the reference signal is distinguishable from the detectable signal and is independent of the enzyme activity of the sample. 
     
     
         18 . The method of  claim 17 , wherein the reference signal is used to normalize the aggregate signal. 
     
     
         19 . The method of  claim 11 , further comprising the step of contacting the cell with a cell dye, wherein the cell dye is selected to provide a dye signal that is distinguishable from the aggregate signal. 
     
     
         20 . The method of  claim 19 , wherein the dye signal is used to normalize the aggregate signal. 
     
     
         21 . The method of  claim 19 , wherein the cell is contacted with the cell dye prior to contacting the cell with the sample. 
     
     
         22 . The method of  claim 19 , wherein the cell is contacted with the cell dye after the cell is contacted with the sample. 
     
     
         23 . The method of  claim 19 , wherein the cell is contacted with the cell dye and the sample during the same time interval. 
     
     
         24 . The method of  claim 11 , wherein the reporting construct does not exhibit useful levels of FRET.