Assays for measuring nucleic acid binding proteins and enzyme activities

Abstract

Processes for measuring DNA or RNA binding proteins, specific nucleic acids, as well as enzyme activities using labeled nucleic acids of labeled protein/peptide molecules are provided.

1 . A method of assaying a sample for an enzyme activity that joins a first substrate with a second substrate to form a product or for a factor that affects the activity of said enzyme, wherein the presence, concentration, or activity of said enzyme or said factor is not known, comprising:
 (a) forming a composition comprising said sample, said first substrate and said second substrate, said first substrate being linked to a luminescent label and said second substrate being linked to a capture moiety;   (b) incubating said composition under conditions wherein said enzyme can form said product at a differing rate in the presence or absence of said enzyme or factor, wherein said product is linked to said luminescent label and said capture moiety and wherein said enzyme or factor is not part of the product;   (c) capturing said capture moiety on an electrode;   (d) applying a voltage to said electrode so as to induce said luminescent label linked to said product to emit luminescence; and   (e) measuring emitted luminescence so as to measure the presence of said enzyme or factor in said sample.   
     
     
         2 . The method of  claim 1 , wherein said enzyme catalyzes formation of a covalent bond between said first substrate and said second substrate. 
     
     
         3 . The method of  claim 1 , wherein said first substrate or said second substrate is a peptide. 
     
     
         4 . The method of  claim 1 , wherein said first substrate or said second substrate is a nucleic acid. 
     
     
         5 . The method of  claim 1 , wherein the luminescent label comprises an electrochemiluminescent label. 
     
     
         6 . The method of  claim 5 , wherein the electrochemiluminescent label comprises ruthenium, osmium or rhenium. 
     
     
         7 . The method of  claim 5 , wherein the electrochemiluminescent label comprises a bipyridyl or phenanthrolyl-containing complex of ruthenium. 
     
     
         8 . The method of  claim 5 , wherein the electrochemiluminescent label comprises RuBpy. 
     
     
         9 . A method of assaying a sample for an enzyme activity that cleaves nucleic acid, comprising:
 a) mixing at least one predetermined single- or double-stranded nucleic acid containing at least one electrochemiluminescent label with a sample, wherein the sample may contain a nucleic acid-cleaving enzyme;   b) incubating the mixture of step (a) under conditions which allow cleavage of the nucleic acid; and   c) analyzing the incubated mixture for cleaved nucleic acid.   
     
     
         10 . A method for assaying a sample for the presence of an enzyme activity that cleaves peptides or proteins, the method comprising:
 a) mixing at least one predetermined peptide or protein containing at least one electrochemiluminescent label with a sample which may contain a peptide or protein-cleaving enzyme;   b) incubating the mixture of step (a) under conditions which allow cleavage of said peptide or protein; and   c) analyzing the incubated mixture for the presence of cleaved peptide or protein.   
     
     
         11 . A method for assaying for the presence of a specific nucleic acid sequence comprising:
 (a) mixing at least one single-stranded nucleic acid sequence which contains a complimentary sequence of said specific nucleic acid sequence, having at least one electrochemiluminescent label, to a sample which may contain said specific nucleic acid sequence;   (b) incubating the mixture of step (a) under conditions which allow the binding of said at least one nucleic acid sequence to said specific nucleic acid sequence to form a duplex;   (c) adding a nucleic acid-cleaving enzyme or reagent to the mixture of step   (b);   (d) incubating the mixture of step (c) under conditions which allow the cleavage of said at least one single-stranded nucleic acid sequence which has not formed a duplex; and   (e) measuring the amount of said duplex to measure said specific nucleic acid sequence.   
     
     
         12 . The method of  claim 11 , wherein the electrochemiluminescent label comprises ruthenium, osmium or rhenium. 
     
     
         13 . The method of  claim 11 , wherein the electrochemiluminescent label comprises a bipyridyl or phenanthrolyl-containing complex of ruthenium. 
     
     
         14 . The method of  claim 11 , wherein the electrochemiluminescent label comprises RuBpy. 
     
     
         15 . The method of  claim 11 , wherein said at least one single-stranded nucleic acid sequence comprises at least one capture moiety. 
     
     
         16 . The method of  claim 15 , wherein the capture moiety is one member of a binding pair selected from the group consisting of: biotin and avidin, biotin and streptavidin, antibody and antigen, receptor and ligand, nucleic acid sequence and complementary nucleic acid sequence. 
     
     
         17 . The method of  claim 11 , wherein prior to step (a), said at least one single-stranded nucleic acid sequence is contacted with a solid phase. 
     
     
         18 . The method of  claim 11 , wherein after step (d), the mixture is contacted with a solid phase.