Methods 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 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 - 20 . (canceled) 
     
     
         21 . A method for screening one or more candidate binding partners for modulating the conformation of 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, wherein a nonlinear-active label is attached to a part of said test molecule that undergoes a conformational change upon interaction with said one or more binding partners;   (b) measuring one or more physical properties of a second-harmonic or sum-frequency light beam emanating from said sample; and   (c) comparing a value of said one or more physical properties measured in step (b) 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, 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 of said test molecule to said one or more candidate binding partners indicates that said one or more candidate binding partners modulate the conformation of said test molecule.   
     
     
         22 . The method of  claim 21 , wherein the conformational change is a local conformational change in the structure of a subpart of the test molecule that occurs upon a binding event with the candidate binding partners. 
     
     
         23 . The method of  claim 21 , wherein the change in the physical properties of a second-harmonic or sum-frequency light beam is not due to rotational motion of the test molecule. 
     
     
         24 . A method for screening one or more candidate binding partners for modulating the conformation of 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, wherein a nonlinear-active label is attached to a part of said test molecule that undergoes a conformational change upon interaction with said one or more binding partners;   (b) measuring one or more physical properties of a second-harmonic or sum-frequency light beam emanating from said sample; and   (c) comparing a value of said one or more physical properties measured in step (b) to a value for said one or more physical properties measured upon binding with a control which is known to bind to the test molecule but not to produce a conformational change or produce a different conformation, 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 upon binding with the control indicates that said one or more candidate binding partners modulate the conformation of said test molecule.   
     
     
         25 . The method of  claim 21 , wherein said test molecule is directly bound to one or more nonlinear-active labels. 
     
     
         26 . The method of  claim 21 , wherein said candidate binding partners are directly bound to one or more nonlinear-active labels. 
     
     
         27 . The method of  claim 21 , wherein the sample in step (a) further comprises a known modulator of said test molecule. 
     
     
         28 . A method for screening one or more candidate binding partners for modulating the conformation of 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, wherein a nonlinear-active label is attached to a part of said test molecule that undergoes a conformational change upon interaction with said one or more binding partners;   (b) measuring one or more physical properties of a second-harmonic or sum-frequency light beam emanating from a sample in step (a);   (c) illuminating a sample in step (a) with one or more light beams at one or more fundamental frequencies, said sample further comprising a known modulator of said test molecule;   (d) measuring one or more physical properties of a second-harmonic or sum-frequency light beam emanating from said a sample in step (c); and   (e) comparing a value of said one or more physical properties measured in step (b) to a value for said one or more physical properties measured in step (d), 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 step (d) indicates that said one or more candidate binding partners modulate the conformation of said test molecule.   
     
     
         29 . The method of  claim 28 , wherein the conformational change is a local conformational change in the structure of a subpart of the test molecule that occurs upon a binding event with the candidate binding partners. 
     
     
         30 . The method of  claim 28 , wherein the change in the physical properties of a second-harmonic or sum-frequency light beam is not due to rotational motion of the test molecule. 
     
     
         31 . The method of  claim 28 , wherein a value of the one or more physical properties measured upon a binding event with the candidate binding partners is compared to a value measure upon binding with a control which is known to bind to the test molecule but not to produce a conformational change. 
     
     
         32 . The method of  claim 28 , wherein said test molecule is directly bound to one or more nonlinear-active labels. 
     
     
         33 . The method of  claim 28 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule. 
     
     
         34 . The method of  claim 28 , wherein said candidate binding partners are directly bound to one or more nonlinear-active labels. 
     
     
         35 . The method of  claim 28 , wherein the sample in step (b) further comprises at least one decorator with nonlinear activity that specifically binds to a candidate binding partner. 
     
     
         36 . The method of  claim 28  wherein the sample in step (b) further comprises at least one decorator with nonlinear activity that specifically binds to a test molecule. 
     
     
         37 . The method of  claim 28  wherein the sample in step (b) further comprises at least one decorator with nonlinear activity that specifically binds to a complex comprising one or more candidate binding partners bound to a test molecule. 
     
     
         38 . The method of  claim 21 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule. 
     
     
         39 . The method of  claim 21 , wherein the sample in step (b) further comprises at least one decorator with nonlinear activity that specifically binds to a candidate binding partner. 
     
     
         40 . The method of  claim 21  wherein the sample in step (b) further comprises at least one decorator with nonlinear activity that specifically binds to a test molecule. 
     
     
         41 . The method of  claim 21  wherein the sample in step (b) further comprises at least one decorator with nonlinear activity that specifically binds to a complex comprising one or more candidate binding partners bound to a test molecule.