Molecular modification assays

Abstract

Systems for detecting molecular modifications and the presence and/or activity of enzymes and/or other agents involved in facilitating or otherwise regulating such modifications.

1 . A method of detecting the activity of an enzyme that operates on an enzyme substrate to form an enzyme product in a sample, comprising:
 contacting the substrate or product with a binding partner that specifically binds to the substrate or to the product but not to both, where the binding partner includes a metal that is involved in binding between the binding partner and the substrate or product;   contacting the substrate with the enzyme;   exposing the sample to light capable of inducing luminescence from the sample; and   measuring a detectable luminescence energy transfer response, without separating the bound substrate or product from the unbound substrate or product, where the detectable luminescence energy transfer response is indicative of the extent of binding between the substrate or product and the binding partner; and   correlating the luminescence energy transfer response with the activity of the enzyme.   
     
     
         2 . The method of  claim 1 , where the metal is selected from the group consisting of aluminum, iron, gallium, europium, and terbium. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , where measuring the detectable luminescence energy transfer response includes measuring luminescence intensity. 
     
     
         6 . The method of  claim 1 , where at least one of the substrate and/or product, and the binding partner includes an energy transfer donor, and at least the other of the substrate and/or product, and the binding partner includes an energy transfer acceptor, such that luminescence energy transfer can occur between the donor and the acceptor. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 6 , where the binding partner includes an energy transfer donor or acceptor, and that energy transfer donor or acceptor includes the metal that is involved in binding between the binding partner and the substrate or product. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 9 , where the energy transfer donor comprises a lanthanide. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 12 , where the energy transfer donor further comprises an organic chelator and a sensitizer moiety. 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 12 , where the lanthanide chelate has the formula 
       
         
           
           
               
               
           
         
         where the lanthanide chelate is associated with the binding partner via a metal-binding functional group, and is optionally further substituted by one or more reactive functional groups or conjugated substances. 
       
     
     
         21 . The method of  claim 1 , where the substrate is a polypeptide, and where the substrate and product are related by a posttranslational modification. 
     
     
         22 . The method of  claim 21 , where the posttranslational modification is phosphorylation or dephosphorylation of the polypeptide and the enzyme is selected from the group consisting of kinases and phosphatases. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 1 , where the substrate is a nucleotide, the substrate and product are related by a cyclization or decyclization of the nucleotide, and the enzyme is selected from the group consisting of cyclases and phosphodiesterases. 
     
     
         26 . The method of  claim 1 , where the substrate is a lipid, lipid precursor or lipid fragment, and the enzyme is selected from the group of lipases and lipid ligases. 
     
     
         27 . The method of  claim 1 , where the substrate includes a phosphorylated polypeptide or a nonphosphorylated polypeptide. 
     
     
         28 . The method of  claim 1 , where the substrate includes a cyckzed nucleotide or a noncyclized nucleotide. 
     
     
         29 . The method of  claim 1 , further comprising:
 contacting the substrate and enzyme with a candidate compound; and   determining the ability of the candidate compound to enhance or inhibit enzyme activity by its effects on the response.   
     
     
         30 . The method of  claim 1 , further comprising:
 providing a sample holder having a plurality of sample sites supporting a corresponding plurality of samples: and   repeating the steps of contacting, detecting, and correlating for the plurality of samples.   
     
     
         31 . A kit for performing the method of  claim 1 , comprising:
 a phosphorylated or nonphosphorylated polypeptide; and   a binding partner that specifically binds to one of the phosphorylated and nonphosphorylated polypeptide but not both, wherein the binding partner includes a metal required for specific binding of the binding partner to the one form of the polypeptide.   
     
     
         32 . A kit for performing the method of  claim 1 , comprising:
 a cyclized or noncyclized form of a nucleotide; and   a binding partner that specifically binds to one of the cyclized form and the noncyclized form of the nucleotide, but not both, wherein the binding partner includes a metal required for specific binding of the binding partner to the one form of the nucleotide.   
     
     
         33 . A method of detecting phosphorylation or nonphosphorylation of a polypeptide in a sample, comprising:
 contacting the polypeptide with a binding partner that specifically binds to the phosphorylated polypeptide or to the nonphosphorylated polypeptide but not to both, where the binding partner includes a metal that is required for binding between the binding partner and the phosphorylated polypeptide or nonphosphorylated polypeptide:   exposing the sample to light capable of inducing luminescence from the sample; and   measuring a detectable luminescence energy transfer, where the luminescence energy transfer is indicative of the extent of binding between the polypeptide and the binding partner without separating the bound polypeptide from the unbound polypeptide; and   correlating the luminescence energy transfer with the extent of phosphorylation or nonphosphorylation of the polypeptide, or with the activity of an enzyme that affects phosphorylation or nonphosphorylation of the polypeptide.   
     
     
         34 . A method of detecting cyclization or noncyclization of a nucleotide in a sample, comprising:
 contacting a nonradioactive nucleotide with a binding partner that specifically binds to a cyclized nucleotide or to a noncyclized nucleotide but not to both, substantially without regard to the nucleoside portion of the nucleotide, where the binding partner includes a metal that is required for binding between the binding partner and the cyclized nucleotide or noncyclized nucleotide;   exposing the sample to capable of inducing luminescence from the sample; and   measuring a detectable luminescence energy transfer, where the luminescence energy transfer is indicative of the extent of binding between the nucleotide and the binding partner; and   correlating the response with the extent of cyclization or noncyclization of the nucleotide, or with the activity of an enzyme that affects cyclization or noncyclization of the nucleotide.