Compositions and methods for detecting protease activity

Abstract

The invention provides a method of determining activity of a protease. The method can include the steps of (a) providing a protease substrate including a protein moiety attached to a nucleic acid moiety and a ligand moiety; (b) contacting the protease substrate with a protease under conditions wherein the protease catalyzes cleavage of the protein moiety, thereby producing a proteolytic product wherein the nucleic acid moiety is separated from at least a portion of the protein moiety and the ligand moiety; (c) contacting the proteolytic product with a receptor under conditions wherein the ligand moiety binds to the receptor to form a complex; (d) separating the complex from the nucleic acid moiety, thereby forming a separation product including the nucleic acid moiety; (e) contacting the separation product with a probe nucleic acid under conditions wherein the nucleic acid moiety hybridizes to a complementary sequence of the probe; and (f) detecting hybridization of the separation product to the probe, thereby determining activity of the protease.

1 - 24 . (canceled)  
   
   
       25 . A protease substrate comprising a molecule having a protein moiety, a nucleic acid moiety and a label moiety, wherein said protein moiety comprises a protease recognition sequence capable of being cleaved by a protease, and wherein said label moiety is attached in a configuration that allows said label moiety to be separated from a portion of said protein moiety upon proteolysis of said protein moiety.  
   
   
       26 . The composition of  claim 25 , wherein said label moiety comprises a ligand.  
   
   
       27 . The composition of  claim 25 , wherein said label moiety is attached to said nucleic acid moiety.  
   
   
       28 . The composition of  claim 25 , wherein said label moiety is attached to said protein moiety  
   
   
       29 . The composition of  claim 25 , wherein said protein moiety comprises a carboxy or amino terminal residue that is attached to said nucleic acid moiety.  
   
   
       30 . The composition of  claim 25 , wherein said protein moiety is attached to the 3′ end or 5′ end of said nucleic acid moiety.  
   
   
       31 . The composition of  claim 25 , wherein said protein moiety comprises an internal residue that is attached to said nucleic acid moiety.  
   
   
       32 . The composition of  claim 25 , wherein said protein moiety is attached to an internal nucleotide of said nucleic acid moiety.  
   
   
       33 . The composition of  claim 25 , wherein attachment of said protein moiety to said nucleic acid moiety is mediated by a linker that comprises said label moiety.  
   
   
       34 . The composition of  claim 25 , further comprising a second label moiety attached to said portion of said protein moiety that is separated from said label moiety upon proteolysis.  
   
   
       35 . The composition of  claim 34 , wherein said label moiety is attached to said nucleic acid moiety and said second label moiety is attached to said protein moiety.  
   
   
       36 . The composition of  claim 34 , wherein said label moiety is attached to a first location of said protein moiety and said second label moiety is attached to a second location of said protein moiety.  
   
   
       37 . A composition comprising a plurality of different protease substrates, wherein each different protease substrates comprises a molecule having a different protein moiety covalently attached to a nucleic acid moiety and a label moiety, wherein the nucleotide sequence of said nucleic acid moiety is unique to the amino acid sequence of said different protein moiety, wherein said protein moiety comprises a protease recognition sequence capable of being cleaved by a protease, and wherein said label moiety is attached in a configuration that allows said label moiety to be separated from a portion of said protein moiety upon proteolysis of said protein moiety.  
   
   
       38 . The composition of  claim 37 , wherein said label moiety is the same for said different protease substrates.  
   
   
       39 . The composition of  claim 37 , wherein said label moiety is different for said different protease substrates.  
   
   
       40 . The composition of  claim 37 , wherein said label moiety comprises a ligand.  
   
   
       41 . The composition of  claim 37 , wherein said label moiety is attached to said nucleic acid moiety.  
   
   
       42 . The composition of  claim 37 , wherein said label moiety is attached to said protein moiety  
   
   
       43 . The composition of  claim 37 , wherein said protein moiety comprises a carboxy or amino terminal residue that is attached to said nucleic acid moiety.  
   
   
       44 . The composition of  claim 37 , wherein said protein moiety is attached to the 3′ end or 5′ end of said nucleic acid moiety.  
   
   
       45 . The composition of  claim 37 , wherein said protein moiety comprises an internal residue that is attached to said nucleic acid moiety.  
   
   
       46 . The composition of  claim 37 , wherein said protein moiety is attached to an internal nucleotide of said nucleic acid moiety.  
   
   
       47 . The composition of  claim 37 , wherein attachment of said protein moiety to said nucleic acid moiety is mediated by a linker that comprises said label moiety.  
   
   
       48 . The composition of  claim 37 , further comprising a second label moiety attached to said portion of said protein moiety that is separated from said label moiety upon proteolysis.  
   
   
       49 . The composition of  claim 48 , wherein said label moiety is attached to said nucleic acid moiety and said second label moiety is attached to said protein moiety.  
   
   
       50 . The composition of  claim 48 , wherein said label moiety is attached to a first location of said protein moiety and said second label moiety is attached to a second location of said protein moiety.  
   
   
       51 . The composition of  claim 48 , wherein said second label moiety is the same for said different protease substrates.  
   
   
       52 . The composition of  claim 48 , wherein said second label moiety is different for said different protease substrates.