Method using a nonlinear optical technique for detection of interactions involving a conformational change

Abstract

A nonlinear optical technique, such as second or third harmonic or sum or difference frequency generation, is used to detect binding interactions, or the degree or extent of binding, that comprise a conformational change. In one aspect of the present invention, the nonlinear optical technique detects a conformational change in a probe due to target binding. In another aspect of the invention, the nonlinear optical technique screens candidate probes by detecting a conformational change due to a probe-target interaction. In another aspect of the invention, the nonlinear optical technique screens candidate modulators of a probe-target interaction by detecting a conformational change in the presence of the modulator.

1 - 195 . (canceled)  
     
     
         196 . A method for screening one or more candidate binding partners for binding to a test molecule comprising: 
 (a) illuminating a sample with one or more light beams at one or more fundamental frequencies, said sample comprising said test molecule exposed to said one or more candidate binding partners; and    (b) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample; wherein a change in the value of said one or more physical properties measured in step (b) relative to a value for said one or more physical properties measured in the absence of exposure of said test molecule to said one or more candidate binding partners indicates that said one or more candidate binding partners bind said test molecule.    
     
     
         197 . The method of  claim 196 , wherein said test molecule is bound to one or more nonlinear-active labels.  
     
     
         198 . The method of  claim 196 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         199 . The method of  claim 196 , wherein said candidate binding partners are bound to one or more nonlinear-active labels.  
     
     
         200 . The method of  claim 196 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         201 . A method for screening one or more candidate modulator molecules for the ability to modulate an interaction between a test molecule and its binding partner comprising: 
 (a) illuminating a sample with one or more light beams at one or more fundamental frequencies, said sample comprising said test molecule exposed to 
 (i) said binding partner, and  
 (ii) said one or more candidate modulator molecules; and  
   (b) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample; wherein a change in the value of said one or more physical properties measured in step (b) relative to the value for said one or more physical properties measured in the absence of exposure to said one or more candidate modulator molecules indicates that said one or more candidate modulator molecules modulate the interaction between said test molecule and its binding partner.    
     
     
         202 . The method of  claim 201 , wherein said test molecule is bound to one or more nonlinear-active labels.  
     
     
         203 . The method of  claim 201 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         204 . The method of  claim 201 , wherein said candidate binding partners are bound to one or more nonlinear-active labels.  
     
     
         205 . The method of  claim 201 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         206 . A method for detecting a conformational change in a test molecule upon binding of the test molecule to a binding partner comprising: contacting said test molecule with one or more candidate binding partners, wherein the test molecule or the one or more candidate binding partners is labeled with a nonlinear-active moiety that is not native to the test molecule or the one or more candidate binding partners, respectively; 
 (a) illuminating said contacted test molecule with one or more light beams at one or more fundamental frequencies; and    (b) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample; wherein a change in the value of said one or more physical properties measured in step (b) relative to the value for said one or more physical properties measured in the absence of said one or more candidate binding partners indicates that at least one of said one or more candidate binding partners bind to said test molecule and that said binding induces a conformational change in said candidate binding partners, in said test molecule, or in both said candidate binding partners and said test molecule.    
     
     
         207 . The method of  claim 206 , wherein said test molecule is bound to one or more nonlinear-active labels.  
     
     
         208 . The method of  claim 206 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         209 . The method of  claim 206 , wherein said candidate binding partners are bound to one or more nonlinear-active labels.  
     
     
         210 . The method of  claim 206 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         211 . A method for detecting the degree or extent of the conformational change induced by binding between a test molecule and one or more candidate binding partners comprising: 
 (a) contacting said test molecule with one or more candidate binding partners, wherein the test molecule or the one or more candidate binding partners is labeled with a nonlinear-active moiety that is not native to the test molecule or the one or more candidate binding partners, respectively;    (b) illuminating said contacted test molecule with one or more light beams at one or more fundamental frequencies; and    (c) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample; wherein the extent of the change in the value of said one or more physical properties measured in step (c) relative to the value for said one or more physical properties measured in the absence of said one or more candidate binding partners indicates the degree or extent of the conformational change that said binding induces.    
     
     
         212 . The method of claim  21   1 , wherein said test molecule is bound to one or more nonlinear-active labels.  
     
     
         213 . The method of claim  21   1 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         214 . The method of claim  21   1 , wherein said candidate binding partners are bound to one or more nonlinear-active labels.  
     
     
         215 . The method of claim  21   1 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.