Fret protease assays for clostridial toxins
AssigneeAllergan, Inc.
InventorsLance E. Steward, Ester Fernandez-Salas, Kei Roger Aoki
Life of this patent
Estimating the term…
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.
Claims26
A botulinum toxin substrate, comprising:
- a) a donor fluorophore;
- b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore, wherein the acceptor is an acceptor fluorophore;
- and
- c) a BoNT/A recognition sequence comprising a BoNT/A P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence, said BoNT/A P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ 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 fluorophore.
A botulinum 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 botulinum toxin recognition sequence comprising a BoNT/A P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence including a BoNT/A P1-P1′ cleavage site, said BoNT/A P1P1′ cleavage site intervening between said donor fluorophore and said acceptor;
- wherein either said donor fluorophore or said acceptor is not positioned within said BoNT/A P5-P4-P3-P2-P1-P1′-P2′-P3′-P4′-P5′ cleavage site sequence;
- and
- wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor.
Dependencies are inferred from the claim text.