New applications of fluorescence polarization for enzyme assays and in genomics

We have developed new, fluorescence polarization-based approaches for performing enzyme assays in homogeneous solutions and for detecting the hybridization of peptide nucleic acids (PNAs) to DNA targets. In the first method, fluorescein-labeled peptides serving as protein kinase sustrates are thiophosphorylated in the presence of the ATP analog ATP gammaS. A sulfur-reactive biotin derivative is then added to the mixture and allowed to react with the thiophosphorylated peptide. The formation of a fluorescein-labeled, biotinylated product can be detected by measuring the fluorescence polarization signal of fluorescein upon addition of streptavidin. In the second method, fluorescein-labeled peptides are subjected to enzymatic phosphorylation, dephosphorylation, or proteolytic cleavage by protein kinases, phosphatases, and proteases. The differential binding of the enzymatic substrates and products to cationic polymers such as polyarginine can be conveniently measured by fluorescence polarization. Finally, we have discovered that the process of hybridization of peptide nucleic acid probes (PNAs) to their target DNA molecules can be followed by measuring the fluorescence polarization of a fluorophore attached to the PNA probes. These measurements can be done either in the presence or absence of polylysine in solution. Examples of the application of this method for single nucleotide polymorphism (SNP) typing are presented.