Use of fluorescence correlation spectroscopy to identify compounds that bind to target species under isothermal denaturing conditions

Abstract

The present invention provides a method for identifying a test compound that binds to a target species. The method includes: incubating at least one test mixture under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent; detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.

What is claimed is:  
     
         1 . A method for identifying a test compound that binds to a target species, the method comprising: 
 incubating at least one test mixture under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent;    detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and    comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions.    
     
     
         2 . The method of  claim 1  wherein the target species is a polypeptide or a polynucleotide.  
     
     
         3 . The method of  claim 2  wherein the target species is a protein.  
     
     
         4 . The method of  claim 1  wherein the compound binds specifically to the target species.  
     
     
         5 . The method of  claim 1  wherein the compound binds to the target species through hydrophobic, covalent, ionic, or hydrogen bonding interactions.  
     
     
         6 . The method of  claim 1  wherein the isothermal denaturation conditions comprise a temperature equal to or about 10° C. more or less than the T m  value of the target species as determined by DSC.  
     
     
         7 . The method of  claim 6  wherein the isothermal denaturation conditions comprise a temperature equal to or up to about 10° C. less than the T m  value of the target species as determined by DSC.  
     
     
         8 . The method of  claim 1  further comprising incubating at least one test compound, at least one target species, and at least one reporter molecule under isothermal denaturing conditions.  
     
     
         9 . The method of  claim 8  wherein the concentrations of the target species and the reporter molecule are of comparable magnitude.  
     
     
         10 . The method of  claim 9  wherein the concentration of the at least one test compound is in at least a 10-fold excess relative to the concentration of the at least one target species.  
     
     
         11 . The method of  claim 1  wherein each test mixture includes one target species.  
     
     
         12 . The method of  claim 1  wherein each test mixture includes at least two test compounds.  
     
     
         13 . The method of  claim 12  wherein each test mixture includes two to ten test compounds.  
     
     
         14 . A high throughput screening method for identifying a test compound that binds to a target species, the method comprising: 
 incubating a plurality of test mixtures under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent;    detecting a denaturation signal of each target species in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and    comparing the denaturation signal of each target species in the presence of at least one test compound with a denaturation signal of the same target species in the absence of the at least one test compound, under the same isothermal denaturing conditions.    
     
     
         15 . The method of  claim 14  wherein the isothermal denaturation conditions comprise a temperature equal to or about 10° C. more or less than the T m  value of the target species as determined by DSC.  
     
     
         16 . The method of  claim 15  wherein the isothermal denaturation conditions comprise a temperature equal to or up to about 10° C. less than the T m  value of the target species as determined by DSC.  
     
     
         17 . The method of  claim 16  wherein each test mixture includes one target species.  
     
     
         18 . The method of  claim 16  wherein each test mixture includes at least two test compounds.  
     
     
         19 . The method of  claim 18  wherein each test mixture includes two to ten test compounds.  
     
     
         20 . A high throughput screening method for identifying a test compound that binds to a protein, the method comprising: 
 incubating a plurality of test mixtures under isothermal denaturing conditions, each test mixture comprising at least one test compound, and at least one protein, wherein the isothermal denaturing conditions are effective to cause at least a portion of the protein to denature to a measurable extent;    detecting a denaturation signal of each protein in the presence of the at least one test compound by a change in the diffusion properties of the target molecule using fluorescence correlation spectroscopy; and    comparing the denaturation signal of each protein in the presence of at least one test compound with a denaturation signal of the same protein in the absence of the at least one test compound under the same isothermal denaturing conditions.    
     
     
         21 . A system for determining whether a test compound is capable of binding a target species under isothermal denaturing conditions, comprising: 
 (a) a compartment comprising a test mixture comprising: 
 (i) at least one test compound; and  
 (ii) at least one target species, wherein the isothermal denaturing conditions are effective to cause at least a portion of the target species to denature to a measurable extent; and  
   (b) means for detecting a denaturation signal of each target species in the presence of at least one test compound by monitoring the diffusion properties of the target molecule using fluorescence correlation spectroscopy, whereby a decrease in the denaturation signal of the target species in the presence of the at least one test compound as compared to a denaturation signal of the same target species in the absence of the at least one test compound under the same isothermal denaturing conditions is an indication of binding between the target species and the at least one test compound.    
     
     
         22 . The system of  claim 21  wherein the target species is a polypeptide or a polynucleotide.  
     
     
         23 . The sytem of  claim 22  wherein the target species is a protein.  
     
     
         24 . The system of  claim 21  wherein the compound binds specifically to the target species.  
     
     
         25 . The system of  claim 21  wherein the compound binds to the target species through hydrophobic, covalent, ionic, or hydrogen bonding interactions.  
     
     
         26 . The system of  claim 21  wherein the isothermal denaturation conditions comprise a temperature equal to or about 10° C. more or less than the T m  value of the target species as determined by differential scanning calorimetry.  
     
     
         27 . The system of  claim 26  wherein the isothermal denaturation conditions comprise a temperature equal to or up to about 10° C. less than the T m  value of the target species as determined by differential scanning calorimetry.  
     
     
         28 . The system of  claim 21  further comprising means for incubating at least one test compound, at least one target species, and at least one reporter molecule under isothermal denaturing conditions.  
     
     
         29 . The system of  claim 28  wherein the concentrations of the target species and the reporter molecule are of comparable magnitude.  
     
     
         30 . The system of  claim 29  wherein the concentration of the at least one test compound is in at least a 10-fold excess relative to the concentration of the at least one target species.  
     
     
         31 . The system of  claim 21  wherein each test mixture includes one target species.  
     
     
         32 . The system of  claim 21  wherein each test mixture includes at least two test compounds.  
     
     
         33 . The system of  claim 32  wherein each test mixture includes two to ten test compounds.