Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding

To quantitatively understand the binding af®nity and target selectivity of small-molecule RNA inter-actions, it is useful to have a rapid, highly repro-ducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low-molecular weight ligands to RNA by monitoring the ¯uorescence of a covalently incorporated ¯uorophore. As a test sys-tem, the ¯uorescence of a pyrene-derivatized HIV-1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside anti-biotics. The binding isotherms thus obtained ®t well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglyco-sides, the binding af®nity correlates with the number of amines, supporting an electrostatic com-pensation model for binding. Furthermore, the ionic strength dependence con®rms that much of the binding energy is electrostatic. Finally, by measur-ing the binding af®nity in the presence of nucleic acid competitors, we con®rm that although amino-glycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub-optimal. We conclude that this newly developed assay can be generalized to measure the binding af®nities and selectivities of a variety of small molecules to a speci®c RNA target