Screen employing fluorescence anisotropy to identify compounds with affinity for nucleic acids

Abstract

The present invention provides methods for screening for bioactive compounds, in particular those that bind to RNA sequences involved in the pathogenesis of disease or in regulation of a physiological function. The methods involve assessing the stability and/or the conformation of an RNA target in the presence and absence of test ligands, and identifying as a ligand any test ligand that causes a measurable change in target RNA stability and/or conformation. In a preferred embodiment, the effect of a ligand on target RNA stability and/or conformation is assessed by measuring the fluorescence polarization of a fluorescently labeled probe.

What is claimed is:  
     
         1 . A method for identifying a ligand that binds a predetermined target RNA sequence, which comprises: 
 (a) selecting as test ligands a plurality of compounds not known to bind to the target RNA sequence;    (b) incubating the target RNA sequence with one or more fluorescently labeled probes in the presence of a test ligand to produce a test combination;    (c) incubating the target RNA sequence with said one or more probes in the absence of a test ligand to produce a control combination;    (d) measuring one or more fluorescence properties of said probe; and    (e) selecting as a ligand any test ligand that causes a measurable change in at least one of the fluorescence properties measured in step (d) in the test combination relative to the control combination, wherein said property reflects the stability and/or conformation of said target RNA.    
     
     
         2 . A method as defined in  claim 1 , further comprising 
 (f) repeating steps (b)-(e) with a plurality of said test ligands until a ligand that binds to the target RNA sequence is identified.    
     
     
         3 . A method as defined in  claim 1 , wherein the fluorescence property measured in step (d) is fluorescence polarization.  
     
     
         4 . A method as defined in  claim 3 , further comprising subjecting the test and control combinations to conditions that denature a detectable fraction of the target RNA sequence in the control combination.  
     
     
         5 . A method as defined in  claim 4 , wherein the subjecting comprises at least one of altering the temperature, altering the salt concentration, adding denaturing compounds, and combinations thereof.  
     
     
         6 . A method as defined in  claim 3 , wherein the target RNA is from about 5 to about 500 nucleotides in length.  
     
     
         7 . A method as defined in  claim 3 , wherein the probe comprises an oligonucleotide that preferentially hybridizes to a predetermined conformation of the target RNA.  
     
     
         8 . A method as defined in  claim 3 , wherein said probe comprises two or more different fluorescent moieties.  
     
     
         9 . A method as defined in  claim 3 , wherein in step (b), said target RNA sequence is incubated with two or more different probes.