Resonance energy transfer assay with cleavage sequence and spacer

摘要

A molecular construct comprises a donor label, an acceptor label, a linker peptide disposed between the donor and the acceptor, the linker having a cleavage site sequence, and a spacer between at least one of (a) the donor and the cleavage site sequence and (b) the acceptor and the cleavage site sequence. Preferably, the construct is selected from the group consisting of CFP-(SGLRSRA)-SNAP-25-(SNS)-YFP, and CFP-(SGLRSRA)-synaptobrevin-(SNS)-YFP. In preferred embodiments, the linker peptide is a substrate of a botulinum neurotoxin selected from the group consisting of synaptobrevin (VAMP), syntaxin and SNAP-25, or a fragment thereof that can be recognized and cleaved by the botulinum neurotoxin. Advantageously, the spacer increases the electronic coupling between the donor label and the acceptor label relative to a corresponding construct without the spacer.

主权项

1. A method of improving sensitivity of energy transfer between a donor label and an acceptor label, comprising:
providing a first construct comprising the donor label and the acceptor label physically coupled through a linker, wherein the linker comprises an amino acid sequence at least 55 amino acids in length and comprising a cleavage site sequence and a flexible spacer sequence, wherein the spacer sequence is interposed between at least one of the group consisting of the donor label and the cleavage site and the acceptor label and the cleavage site; contacting the first construct with a sample, wherein the sample comprises an activity directed to the cleavage site; detecting a fluorescene emission from the first construct, thereby characterizing an energy transfer between the donor label and the acceptor label, wherein the energy transfer is enhanced relative to that of a second construct that does not include a spacer sequence.