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 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 comprising:
 (a) contacting a ligand to a molecule, wherein the molecule is attached to a nonlinear-active label;   (b) illuminating the molecule; and   (c) detecting that the ligand has bound to the molecule.   
     
     
         22 . The method of  claim 21 , wherein the molecule is a biomolecule. 
     
     
         23 . The method of  claim 21 , wherein the molecule is a receptor. 
     
     
         24 . The method of  claim 21 , wherein the ligand is a drug candidate. 
     
     
         25 . The method of  claim 21 , wherein detecting that the ligand has bound to the molecule comprises detecting that the molecule has undergone a conformational change. 
     
     
         26 . The method of  claim 25 , wherein the conformational change is detected by measuring a second-harmonic or sum-frequency light beam. 
     
     
         27 . The method of  claim 25 , further comprising comparing a value of one or more physical properties measured upon binding of the ligand to the molecule to a value of the physical property measured upon binding of a control to the molecule, wherein the molecule does not undergo the conformational change upon binding the control. 
     
     
         28 . The method of  claim 21 , wherein the molecule is illuminated with a light beam at a fundamental frequency. 
     
     
         29 . The method of  claim 21 , wherein the molecule is a G protein coupled receptor. 
     
     
         30 . The method of  claim 21 , wherein the molecule is bound to a substrate. 
     
     
         31 . The method of  claim 21 , wherein the molecule is attached to a bead. 
     
     
         32 . The method of  claim 21 , wherein the molecule is immobilized in a flow cell. 
     
     
         33 . A method of diagnosing a subject, the method comprising:
 (a) obtaining a sample from the subject, wherein the sample comprises a ligand;   (b) contacting the sample with a molecule, wherein the molecule is attached to a nonlinear-active label;   (c) illuminating the sample;   (d) detecting that the ligand has bound to the molecule; and   (e) diagnosing the subject based on a result of (d).   
     
     
         34 . The method of  claim 33 , wherein the molecule is a biomolecule. 
     
     
         35 . The method of  claim 33 , wherein the molecule is a receptor. 
     
     
         36 . The method of  claim 33 , wherein the molecule is a G protein coupled receptor. 
     
     
         37 . The method of  claim 33 , wherein the detecting that the ligand has bound to the molecule comprises detecting that the molecule has undergone a conformational change. 
     
     
         38 . The method of  claim 37 , wherein the conformational change is detected by measuring a second-harmonic or sum-frequency light beam. 
     
     
         39 . The method of  claim 37 , further comprising comparing a value of one or more physical properties measured upon binding of the ligand to the molecule to a value of the physical property measured upon binding of a control to the molecule, wherein the molecule does not undergo the conformational change upon binding the control. 
     
     
         40 . The method of  claim 33 , wherein the sample is illuminated with a light beam at a fundamental frequency. 
     
     
         41 . The method of  claim 33 , further comprising providing a treatment to the subject based on the diagnosis. 
     
     
         42 . The method of  claim 33 , wherein the subject is diagnosed as having a risk factor for a disease. 
     
     
         43 . The method of  claim 33 , wherein the subject is diagnosed as having a disease. 
     
     
         44 . The method of  claim 33 , wherein the ligand is a biological component. 
     
     
         45 . The method of  claim 33 , wherein the ligand is a virus. 
     
     
         46 . The method of  claim 33 , wherein the ligand is a cell. 
     
     
         47 . The method of  claim 33 , wherein the ligand is a first nucleic acid. 
     
     
         48 . The method of  claim 47 , wherein the molecule is a second nucleic acid, and wherein the binding is a hybridization. 
     
     
         49 . The method of  claim 33 , wherein the sample is contacted with an array of molecules. 
     
     
         50 . The method of  claim 33 , wherein the molecule is bound to a substrate. 
     
     
         51 . The method of  claim 33 , wherein the molecule is attached to a bead. 
     
     
         52 . The method of  claim 33 , wherein the molecule is immobilized in a flow cell. 
     
     
         53 . A method of evaluating a degree of binding of a ligand to a molecule, the method comprising:
 (a) contacting the ligand with the molecule, wherein the molecule is attached to a nonlinear-active label;   (b) illuminating the molecule;   (c) measuring a value of a second-harmonic or sum-frequency light beam emanating from the molecule;   (d) determining a change in the value relative to a control value; and   (e) correlating the change to the degree of binding of the ligand to the molecule.   
     
     
         54 . The method of  claim 53 , wherein the molecule is a biomolecule. 
     
     
         55 . The method of  claim 53 , wherein the molecule undergoes a conformational change upon binding to the molecule by the ligand. 
     
     
         56 . The method of  claim 55 , wherein the control value is measured upon binding of a control to the molecule, wherein the molecule does not undergo a conformational change upon binding the control. 
     
     
         57 . The method of  claim 53 , wherein the molecule is illuminated with a light beam at a fundamental frequency. 
     
     
         58 . The method of  claim 53 , wherein the molecule is a first nucleic acid. 
     
     
         59 . The method of  claim 58 , wherein the ligand is a second nucleic acid. 
     
     
         60 . The method of  claim 59 , wherein the binding is a hybridization and the degree of binding is a degree of hybridization. 
     
     
         61 . The method of  claim 53 , wherein the correlating identifies a single nucleotide polymorphism between the ligand and the molecule. 
     
     
         62 . The method of  claim 53 , wherein the ligand is taken from a subject, and the method further comprises diagnosing the subject based on the correlating. 
     
     
         63 . The method of  claim 62 , further comprising providing a treatment to the subject based on the diagnosis. 
     
     
         64 . The method of  claim 53 , wherein the molecule is bound to a substrate. 
     
     
         65 . The method of  claim 53 , wherein the molecule is attached to a bead. 
     
     
         66 . The method of  claim 53 , wherein the molecule is immobilized in a flow cell. 
     
     
         67 . A method for detecting binding of a biological component to a molecule, the method comprising:
 (a) contacting the biological component with the molecule, wherein the molecule is attached to a nonlinear-active label;   (b) illuminating the molecule; and   (c) detecting that the molecule has undergone a conformational change.   
     
     
         68 . The method of  claim 67 , wherein the molecule is a biomolecule. 
     
     
         69 . The method of  claim 67 , wherein the biological component is a virus. 
     
     
         70 . The method of  claim 67 , wherein the biological component is a cell. 
     
     
         71 . The method of  claim 67 , wherein the molecule undergoes the conformational change upon binding to the molecule by the biological component. 
     
     
         72 . The method of  claim 71 , further comprising comparing a value of one or more physical properties measured upon binding of the biological component to the molecule to a value of the physical property measured upon binding of a control to the molecule, wherein the molecule does not undergo the conformational change upon binding the control. 
     
     
         73 . The method of  claim 67 , wherein the molecule is illuminated with a light beam at a fundamental frequency. 
     
     
         74 . The method of  claim 67 , wherein the conformational change is detected by measuring a second-harmonic or sum-frequency light beam. 
     
     
         75 . The method of  claim 74 , further comprising correlating the measurement to a degree of binding of the biological component to the molecule. 
     
     
         76 . The method of  claim 67 , wherein the molecule is bound to a substrate. 
     
     
         77 . The method of  claim 67 , wherein the molecule is attached to a bead. 
     
     
         78 . The method of  claim 67 , wherein the molecule is immobilized in a flow cell.