Detection of binding factors with fluorescence polarization

Abstract

This invention relates to a simple and quick method for the detection, identification and/or quantitation of binding factors using fluorescence techniques. A fluorescent probe is incubated with a factor or group of factors, and the presence of a factor capable of binding the probe can be detected by fluorescence polarization. When coupled with a separation step, this invention allows on-line monitoring of binding complex formation.

We claim:  
     
         1 . A method for detecting a binding factor for a probe, comprising: 
 (a) labeling the probe with a fluorophore;    (b) incubating the labeled probe with a factor or a group of factors which may bind the labeled probe to form a binding complex;    (c) separating the binding complex and the free probe into different fractions; and    (d) subjecting each fraction from step (c) to fluorescence polarization measurement under conditions wherein the binding complex produces a fluorescence pattern different from that of the free probe, thereby allowing detection of the binding complex.    
     
     
         2 . The method of  claim 1  wherein the free probe and the complex are separated by using capillary electrophoresis.  
     
     
         3 . The method of  claim 1  wherein the group of factors comprises a chemical compound library.  
     
     
         4 . The method of  claim 4  wherein the chemical compound library is a combinatorial library.  
     
     
         5 . The method of  claim 1  wherein the group of factors comprises a mixture of natural products.  
     
     
         6 . The method of  claim 6  wherein the mixture of natural products comprises a cell lysate.  
     
     
         7 . The method of  claim 1  wherein the group of factors comprises nucleic acid.  
     
     
         8 . The method of  claim 7  wherein the nucleic acid is genomic DNA.  
     
     
         9 . The method of  claim 8  wherein the probe is capable of binding to modified DNA.  
     
     
         10 . The method of  claim 10  wherein the modified DNA is a DNA adduct.  
     
     
         11 . The method of  claim 1  wherein the probe is selected from the group consisting of protein and nucleic acid.  
     
     
         12 . The method of  claim 1  wherein the probe has a molecular weight of less than about 10,000 daltons.  
     
     
         13 . The method of  claim 1  wherein the probe has a molecular weight of less than about 5,000 daltons.  
     
     
         14 . The method of  claim 1  wherein the probe has a molecular weight of less than about 3,000 daltons.  
     
     
         15 . The method of  claim 1  further comprising the step of determining binding affinity and/or stoichiometry between the probe and the binding factor.  
     
     
         16 . The method of  claim 1  wherein the fluorophore is fluorescein.  
     
     
         17 . A method for detecting a nucleic acid damage in a nucleic acid sample, comprising: 
 (a) incubating the sample with 
 (i) a polypeptide which is capable of binding the damaged nucleic acid; and  
 (ii) a fluorophore-labeled probe which is capable of forming a complex with the polypeptide to compete with the damaged nucleic acid for the polypeptide; and  
   (b) analyzing the incubation mixture under conditions wherein the complex formed between the probe and the polypeptide produces a fluorescence pattern different from that of a free probe.    
     
     
         18 . The method of  claim 17  wherein the polypeptide is an antibody which is capable of binding damaged DNA.  
     
     
         19 . The method of  claim 17  wherein the DNA damage is a covalent modification.  
     
     
         20 . The method of  claim 17  wherein the DNA damage is a benzopyrene addition.  
     
     
         21 . The method of  claim 17  wherein the DNA sample is genomic DNA.  
     
     
         22 . A method for detecting a fluorophore labeled probe, comprising: 
 (a) incubating a probe with a fluorophore under conditions which allow labeling of the probe by the fluorophore; and    (b) subjecting the incubation mixture to fluorescence polarization under conditions wherein the fluorophore labeled probe produces a fluorescence pattern which is different from that of a free probe which is not labeled by the fluorophore.    
     
     
         23 . The method of  claim 22  further comprising the step of fractionating the incubation mixture.