Methods and Compositions for Detecting Botulinum Neurotoxin

Abstract

The present invention provides a molecular construct capable of fluorescent resonance energy transfer (FRET), comprising a linker peptide, and donor and acceptor fluorophore moieties, where the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin, syntaxin and SNAP-25, or a fragment thereof capable being cleaved by the botulinum neurotoxin, and separates the donor and acceptor fluorophores by a distance of not more than 10 nm, and where emission spectrum of the donor fluorophore moiety overlaps with the excitation spectrum of the acceptor fluorophore moiety; or where the emission spectra of the fluorophores are detectably different. Also provided are isolated nucleic acid expressing the construct, kits comprising said construct and cell lines comprising said nucleic acid. Further provided are methods of detecting a BoNT using the above described construct via FRET, and methods for detecting a BoNT using surface plasmon resonance imaging.

We claim: 
     
         1 . A molecular construct capable of fluorescent resonance energy transfer (FRET), comprising a linker peptide, a donor fluorophore moiety and an acceptor fluorophore moiety, wherein the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin, syntaxin and SNAP-25, or a fragment thereof that is able to be cleaved by the botulinum neurotoxin, and separates the donor and acceptor fluorophores by a distance of not more than 10 nm, and wherein emission spectrum of the donor fluorophore moiety overlaps with the excitation spectrum of the acceptor fluorophore moiety. 
     
     
         2 . The construct of  claim 1 , wherein the donor fluorophore moiety is a green fluorescent protein or a variant thereof, and the acceptor fluorophore moiety is a corresponding variant of the green fluorescent protein. 
     
     
         3 . The construct of  claim 1 , wherein the linker peptide comprises at least about 14 amino acid residues and an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. 
     
     
         4 . The construct of  claim 3 , wherein the linker peptide comprises at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, or 25 amino acid residues. 
     
     
         5 . The construct of  claim 3 , wherein the linker peptide comprises at least about 30 amino acid residues. 
     
     
         6 . The construct of  claim 3 , wherein the linker peptide comprises at least about 35, 40, 45, 50, 55, 60, 65, or 70 amino acid residues. 
     
     
         7 . A kit comprising the construct of  claim 1 , and a suitable container. 
     
     
         8 . A method for detecting a botulinum neurotoxin, the method comprising,
 a) providing the construct of  claim 1 , wherein the linker is a substrate protein or a cleavable fragment thereof corresponding to the botulinum neurotoxin to be detected;   b) exposing the construct to a sample suspected of containing a botulinum neurotoxin under a condition under which the botulinum neurotoxin cleaves the protein substrate or a fragment thereof; and   c) detecting and comparing the FRET signal before and after the construct is exposed to the sample,   wherein a decrease in FRET indicates the presence of botulinum neurotoxin in the sample.   
     
     
         9 . The method of  claim 8 , further comprising providing Zn 2+  to the sample to be detected. 
     
     
         10 . The method of  claim 8 , wherein the botulinum neurotoxin is BoNT/A, E, or C, and the corresponding substrate protein is SNAP-25 or a cleavable fragment thereof. 
     
     
         11 . The method of  claim 8 , wherein the botulinum neurotoxin is BoNT/B, D, F, or G, and the corresponding substrate protein is synaptobrevin (Syb) or a cleavable fragment thereof. 
     
     
         12 . The method of  claim 8 , wherein the botulinum neurotoxin is BoNT/C, and the corresponding substrate protein is SNAP-25 or a cleavable fragment thereof. 
     
     
         13 . The method of  claim 8 , wherein the FRET is detected by a method selected from the group consisting of (a) measuring fluorescence emitted at the acceptor (A) emission wavelength and donor (D) emission wavelength and determining energy transfer by the ratio of the respective emission amplitudes; (b) measuring fluorescence lifetime of D; (c) measuring photobleaching rate of D; (d) measuring anisotropy of D or A; and (e) measuring the Stokes shift monomer/excimer fluorescence. 
     
     
         14 . The method of  claim 8 , wherein the fluorophore pair is CFP-YFP. 
     
     
         15 . The method of  claim 8 , wherein the construct is a recombinant protein expressed in a cell. 
     
     
         16 . A method for screening for an inhibitor of a botulinum neurotoxin, comprising
 a) providing a cell genetically engineered to express the construct of  claim 2 , wherein the linker in the construct is a substrate peptide corresponding to the botulinum toxin;   b) exposing said cell to the botulinum neurotoxin in the presence of a candidate inhibitor compound; and   c) detecting FRET signals of the cell before and after said exposing to the botulinum toxin,   wherein an observation of substantially no decrease in FRET, as compared to a cell exposed to the botulinum neurotoxin in the absence of the candidate inhibitor, indicates that the candidate inhibitor is capable of inhibiting the botulinum neurotoxin.   
     
     
         17 . The method of  claim 16 , wherein the candidate compound is among a library of compounds and the method is a high throughput method. 
     
     
         18 . A method for detecting a botulinum neurotoxin, the method comprising the steps of:
 a) depositing a layer of a BoNT target peptide onto a metal surface;   b) exposing said metal surface having BoNT target peptide on its surface to a sample suspected of containing a corresponding BoNT, under conditions to allow the BoNT to cleave the target peptide on the metal surface; and   c) measuring any decrease in the molecular weight of the target peptide bound to the metal surface as a result of BoNT cleavage via surface Plasmon resonant imaging.   
     
     
         19 . The construct of  claim 1 , wherein the linker peptide comprises at least 71 amino acid residues and a sequence selected from the group consisting of SEQ ID NOs: 1-6, and wherein the linker peptide adopts a conformation with which FRET occurs between the donor and acceptor fluorophores. 
     
     
         20 . The construct of  claim 19 , wherein the linker peptide is a full-length molecule of synaptobrevin, syntaxin or SNAP-25. 
     
     
         21 . A method for detecting a botulinum neurotoxin, the method comprising,
 a) providing the construct of  claim 19 ,   b) exposing the construct to a sample suspected of containing a botulinum neurotoxin under a condition under which the botulinum neurotoxin cleaves the protein substrate or a fragment thereof, and   c) detecting and comparing the FRET signal before and after the construct is exposed to the sample,   wherein a decrease in FRET indicates the presence of botulinum neurotoxin in the sample.   
     
     
         22 . A molecular construct comprising a linker peptide, a first fluorophore moiety, and a second fluorophore moiety, wherein the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin, syntaxin and SNAP-25, or a fragment thereof that is able to be cleaved by the botulinum neurotoxin, and wherein emission spectrum of the first fluorophore moiety is detectably different from the excitation spectrum of the second fluorophore moiety. 
     
     
         23 . The construct of  claim 22 , wherein the linker peptide is a full-length molecule of synaptobrevin, syntaxin or SNAP-25. 
     
     
         24 . The construct of  claim 22 , which is anchored to a vesicle. 
     
     
         25 . A method for detecting a botulinum neurotoxin, the method comprising,
 a) providing the construct of  claim 22 ;   b) exposing the construct to a sample suspected of containing a botulinum neurotoxin under a condition under which the botulinum neurotoxin cleaves the protein substrate or a fragment thereof; and   c) detecting spatial separation of the fluorescence signals of the first and second fluorophores,
 wherein occurrence of spatial separation indicates the presence of botulinum neurotoxin in the sample. 
   
     
     
         26 . The method of  claim 25 , wherein the vesicle is inside a live cell, wherein the linker peptide is CFP-SNAP-25 (1-197) linked to SNAP-25 (198-206)YFP, and wherein detection of CFP fluorescence but not YFP fluorescence indicates the existence of presence of botulinum neurotoxin in the sample.