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 detecting a conformational change upon binding of a test molecule to a candidate binding partner in a sample comprising (a) contacting the test molecule with the candidate binding partner; (b) illuminating the contacted test molecule with a light beam at a fundamental frequency; (c) detecting the conformational change by measuring a physical property of a second-harmonic or sum-frequency light beam emanating from the sample. 
     
     
         22 . The method of  claim 21 , further comprising selected a candidate binding partner based on the value of the physical property measured in step (c) relative to the control value. 
     
     
         23 . The method of  claim 21 , wherein a change in the value of the physical property measured in step (c) relative to a control value indicates that the candidate binding partner modulates the conformation of the test molecule. 
     
     
         24 . The method of  claim 21 , wherein the test molecule is contacted with the candidate binding partner at an interface. 
     
     
         25 . The method of  claim 24 , wherein the interface is a lipid bilayer. 
     
     
         26 . The method of  claim 21 , wherein the candidate binding partner is attached to an interface. 
     
     
         27 . A method for detecting a shift in average label angle of 5 degrees or less in a nonlinear-active label upon binding of the test molecule to a candidate binding partner in a sample comprising (a) contacting the test molecule with the candidate binding partner; (b) illuminating the contacted test molecule with a light beam at a fundamental frequency; (c) detecting the shift by measuring a physical property of a second-harmonic or sum-frequency light beam emanating from the sample. 
     
     
         28 . A method for screening a candidate conformation blocker of a test molecule comprising (a) contacting the test molecule with a known binding partner and a candidate conformation blocker, wherein the test molecule undergoes a conformational change upon the binding with the known binding partner; (b) illuminating the contacted test molecule with a light beam at a fundamental frequency; (c) detecting the conformational change by measuring a physical property of a second-harmonic or sum-frequency light beam emanating from the sample. 
     
     
         29 . The method of  claim 28 , wherein an absence of a change in the value of the physical property measured in step (c) relative to a control value indicates that the candidate binding partner blocks the conformation of said test molecule. 
     
     
         30 . The method of  claim 28 , further comprising selecting a candidate conformation blocker based on the value of the physical property measured in step (c) relative to the control value. 
     
     
         31 . The method of  claim 28 , wherein the test molecule is contacted with the candidate binding partner at an interface. 
     
     
         32 . The method of  claim 31 , wherein the interface is a lipid bilayer. 
     
     
         33 . The method of  claim 28 , wherein the candidate conformation blocker is attached to an interface. 
     
     
         34 . A method for screening different types of binding partners of a test molecule in a sample comprising (a) contacting the test molecule with the binding partners; (b) illuminating the contacted test molecule with a light beam at a fundamental frequency; (c) measuring a physical property of a second-harmonic or sum-frequency light beam emanating from the sample; (d) determining the degree or extent of the binding between the test molecule and the binding partners based on the value of the physical property measured in step (c) thereby distinguishing different types of binding partners, wherein at least one of the binding partners induces a conformational change upon binding of a test molecule. 
     
     
         35 . The method of  claim 34 , further comprising selecting a candidate binding partner based on the amount of change in the value of the physical property measured in step (c) relative to a control value. 
     
     
         36 . The method of  claim 34 , wherein the amount of change in the value of the physical property measured in step (c) relative to a control value indicates the degree or extent of the binding between the test molecule and the binding partners. 
     
     
         37 . The method of  claim 34 , wherein the binding partner is an inhibitor. 
     
     
         38 . The method of  claim 34 , wherein the test molecule is contacted with the modulators at an interface. 
     
     
         39 . The method of  claim 38 , wherein the interface is a lipid bilayer. 
     
     
         40 . The method of  claim 34 , wherein the binding partners are attached to an interface. 
     
     
         41 . The method of  claim 21 , wherein the test molecule is a protein. 
     
     
         42 . The method of  claim 21 , wherein the candidate binding partner is a protein. 
     
     
         43 . The method of  claim 21 , wherein a nonlinear-active label is attached to the test molecule, the binding partner, or the interface. 
     
     
         44 . 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. 
     
     
         45 . 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. 
     
     
         46 . The method of  claim 21 , wherein the sample further comprises a modulator molecule. 
     
     
         47 . The method of  claim 21 , wherein the sample further comprises an enhancer. 
     
     
         48 . The method of  claim 47 , where the enhancer is attached to the interface or the test molecule. 
     
     
         49 . The method of  claim 47 , where the enhancer is attached to the binding partner. 
     
     
         50 . The method of  claim 21 , wherein the control value is a value for the physical property measured in the absence of exposure of the test molecule to the candidate binding partner. 
     
     
         51 . The method of  claim 21 , wherein the control value is a value for the physical property measured upon binding with a control binding partner which is known to bind to the test molecule but not to produce a conformational change. 
     
     
         52 . The method of  claim 21 , wherein the test molecule undergoes a conformational change upon binding with the first binding partner. 
     
     
         53 . The method of  claim 21 , wherein the binding partner undergoes a conformational change upon binding with the first binding partner. 
     
     
         54 . The method of  claim 21 , wherein the test molecule is labeled with a nonlinear-active label. 
     
     
         55 . The method of  claim 54 , wherein the nonlinear-active label is not native to the test molecule. 
     
     
         56 . The method of  claim 21 , wherein the binding partner is labeled with a nonlinear-active label. 
     
     
         57 . The method of  claim 56 , wherein the nonlinear-active label is not native to the binding partner. 
     
     
         58 . The method of  claim 21 , wherein the interface is labeled with a nonlinear-active label. 
     
     
         59 . The method of  claim 21 , wherein the nonlinear-active label is a molecular beacon analogue. 
     
     
         60 . The method of  claim 59 , wherein the molecular beacon analogue comprises the nonlinear-active label and an enhancer. 
     
     
         61 . The method of  claim 21 , wherein the sample further comprises a decorator. 
     
     
         62 . The method of  claim 61 , wherein the decorator is attached to the nonlinear-active label. 
     
     
         63 . The method of  claim 21 , further comprising applying an external force field to the sample. 
     
     
         64 . The method of  claim 63 , wherein the external force field is electrical field. 
     
     
         65 . The method of  claim 21 , wherein the external force field is electrical field.