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 identifying a molecule, the method comprising:
 (a) providing a first molecule in a region of an array; and   (b) using an anisotropically emitted nonlinear light beam to identify the first molecule upon an interaction with a second molecule or particle, wherein the first molecule or the second molecule or particle comprises a nonlinear-active portion capable of producing the anistropically emitted nonlinear light beam in response to illumination by a fundamental light beam.   
     
     
         22 . The method of  claim 21 , wherein the molecule is located at an interface. 
     
     
         23 . The method of  claim 22 , wherein the interface is a lipid bilayer. 
     
     
         24 . The method of  claim 21 , wherein the molecule is located in a bulk phase. 
     
     
         25 . The method of  claim 24 , further comprising poling the molecule by an electric field. 
     
     
         26 . The method of  claim 21 , wherein the first molecule and the second molecule or particle comprise nonlinear-active portions capable of producing the anistropically emitted nonlinear light beam in response to illumination by a fundamental light beam. 
     
     
         27 . The method of  claim 21 , wherein the interaction is a binding event. 
     
     
         28 . The method of  claim 21 , wherein the anisotropically emitted nonlinear light beam is at a sum or difference frequency. 
     
     
         29 . A method for identifying a molecule, the method comprising:
 (a) providing a first molecule in a region of an array; and   (b) using a coherent light beam to identify the first molecule upon an interaction with a second molecule or particle, wherein the first molecule or the second molecule or particle comprises a portion capable of producing the coherent light beam in response to illumination by a fundamental light beam, and wherein the fundamental light beam and the coherent light beam have well-defined phase relationships.   
     
     
         30 . The method of  claim 29 , wherein the first molecule and the second molecule or particle comprise a portion capable of producing the coherent light beam in response to illumination by a fundamental light beam 
     
     
         31 . The method of  claim 29 , further comprising providing illumination by at least one additional fundamental light beam. 
     
     
         32 . An apparatus for probing of a conformational change of a molecule, comprising:
 a non-centrosymmetric region containing the molecule; and   a system for detecting a nonlinear light beam generated by a nonlinear-active portion of the molecule in response to the fundamental light beam provided from a light source, said nonlinear light beam being time-dependent on the conformational change of the molecule.   
     
     
         33 . The apparatus of  claim 32 , wherein the nonlinear light beam is coherent. 
     
     
         34 . The apparatus of  claim 33 , wherein the fundamental light beam and the nonlinear light beam have well-defined phase relationships. 
     
     
         35 . The apparatus of  claim 32 , wherein the nonlinear light beam is at a sum or difference frequency. 
     
     
         36 . The apparatus of  claim 32 , wherein the non-centrosymmetric region is a region of an array. 
     
     
         37 . A method for measuring a dipole moment change during interaction of a test molecule with a candidate binding partner, the method comprising:
 (a) exposing, in a sample region, the test molecule to the candidate binding partner; and   (b) measuring the dipole moment change of the test molecule or the candidate binding partner using a nonlinear optical technique.   
     
     
         38 . The method of  claim 37 , further comprising measuring the dipole moment change of the test molecule and the candidate binding partner. 
     
     
         39 . The method of  claim 37 , further comprising measuring the interaction in real time. 
     
     
         40 . The method of  claim 37 , wherein the dipole moment change is accompanied by a conformational change. 
     
     
         41 . The method of  claim 40 , further comprising measuring the conformational change. 
     
     
         42 . The method of  claim 40 , wherein the interaction is a binding event. 
     
     
         43 . The method of  claim 42 , further comprising comparing the dipole moment change measured upon the binding event to a dipole moment change measured upon binding with a control which is known to bind the test molecule but not to produce a conformational change. 
     
     
         44 . The method of  claim 37 , further comprising exposing the test molecule to the candidate binding partner at an interface. 
     
     
         45 . The method of  claim 44 , wherein the interface is a lipid bilayer. 
     
     
         46 . The method of  claim 37 , wherein the candidate binding partner is attached to an to an interface. 
     
     
         47 . A method for measuring a conformational change during interaction of a test molecule with a candidate binding partner, the method comprising:
 (a) exposing, in a non-centrosymmetric region of a sample, the test molecule to the candidate binding partner; and   (b) measuring the conformational change of the test molecule or the candidate binding partner using a nonlinear light beam generated by one or more nonlinear-active portions of the test molecule or the binding partner in response to a fundamental light beam transmitted or reflected through the sample.   
     
     
         48 . The method of  claim 47 , further comprising measuring the conformational change of the test molecule and the candidate binding partner. 
     
     
         49 . The method of  claim 47 , further comprising using a nonlinear light beam generated by one or more nonlinear-active portions of the test molecule and the binding partner. 
     
     
         50 . The method of  claim 47 , further comprising measuring the interaction in real time. 
     
     
         51 . The method of  claim 47 , wherein the interaction is a binding event. 
     
     
         52 . The method of  claim 51 , further comprising comparing the conformational change measured upon the binding event to a conformational change measured upon binding with a control which is known to bind the test molecule but not to produce a conformational change. 
     
     
         53 . The method of  claim 47 , further comprising exposing the test molecule to the candidate binding partner at an interface. 
     
     
         54 . The method of  claim 53 , wherein the interface is a lipid bilayer. 
     
     
         55 . The method of  claim 47 , wherein the candidate binding partner is attached to an to an interface. 
     
     
         56 . The method of  claim 47 , 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 partner. 
     
     
         57 . A method for detecting a molecule, the method comprising:
 (a) providing a sample having a non-centrosymmetric region containing the molecule, wherein the molecule comprises a nonlinear-active portion; and   (b) detecting the molecule using a nonlinear optical response of the nonlinear-active portion of the molecule to a fundamental light beam transmitted or reflected through the sample.   
     
     
         58 . A method for measuring an interaction of a ligand with a molecule, the method comprising:
 (a) adding the ligand to a non-centrosymmetric region of a sample, said non-centrosymmetric region containing the molecule; and   (b) measuring the interaction of the molecule using a nonlinear light beam generated at the non-centrosymmetric region in response to a fundamental light beam.   
     
     
         59 . The method of  claim 58 , wherein said molecule is nonlinear-active. 
     
     
         60 . The method of  claim 58 , wherein said molecule is labeled with a nonlinear-active moiety. 
     
     
         61 . The method of  claim 58 , wherein the interaction is accompanied by structural change of the molecule. 
     
     
         62 . The method of  claim 58 , wherein the nonlinear light beam is generated by one or more nonlinear-active portions of the molecule or the ligand. 
     
     
         63 . The method of  claim 61 , wherein the structural change is a local structural change in the structure of a subpart of the molecule that occurs upon a binding event with the ligand.