Fret protease assays for clostridial toxins
AssigneeAllergan, Inc.
InventorsLance E. Steward, Ester Fernandez-Salas, Kei Roger Aoki
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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.
Claims95
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 cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype A (BoNT/A) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/A recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype B (BoNT/B) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/B recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype C1 (BoNT/C1) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/C1 recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype D (BoNT/D) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/D recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype E (BoNT/E) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/E recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype F (BoNT/F) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/F recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A botulinum toxin serotype G (BoNT/G) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a BoNT/G recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A tetanus toxin (TeNT) substrate, comprising:
- (a) a donor fluorophore;
- (b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore;
- and
- (c) a TeNT recognition sequence comprising a cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
A method of determining clostridial toxin protease activity, comprising the steps of:
- (a) treating a sample, under conditions suitable for clostridial toxin protease activity, with a 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 cleavage site,
- wherein said cleavage site intervenes between said donor fluorophore and said acceptor and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor;
- (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.
Dependencies are inferred from the claim text.