FRET Assay for Ligands Targeting the Bacterial A-Site RNA.

A robust, fluorescence-based analysis and discovery platform is described for bacterial A-site binders. The assay relies on an incorporated isomorphic fluorescent uridine analog, which substitutes the A-site's U1406 and serves as a FRET donor to an A-site bound coumarin-labeled aminoglycoside that serves as the FRET acceptor. Binding efficiency of unlabeled A-site ligands can be determined by competition experiments, where the acceptor-labeled aminoglycoside is displaced. The replacement efficiency is gauged by the concentration-dependent loss of the sensitized FRET acceptor's signal with concomitant restoration of the donor's emission. Plotting the relative emission intensity of both the donor and acceptor as a function of ligand concentration followed by fitting of the data points to a dose-response curve yields IC50 values, one possible measure of the antibiotic potency of new A-site binders.