Rapid kinetic characterization of hammerhead ribozymes by real-time monitoring of fluorescence resonance energy transfer (FRET)

In established methods for analyzing ribozyme kinetics, radiolabeled RNA su bstrates are primarily used. Each data point requires the cumbersome sampli ng, gel electrophoretic separation, and quantitation of reaction products, apart from the continuous loss of substrate by radioactive decay. We have u sed stable, double fluorescent end-labeled RNA substrates. Fluorescence of one fluorophore is quenched by intramolecular energy transfer (FRET), Upon substrate cleavage, both dyes become separated in two RNA products and fluo rescence is restored, This can be followed in real time and ribozyme reacti ons can be analyzed under multiple (substrate excess) and under single (rib ozyme excess) turnover conditions, A detailed comparison of unlabeled, sing le, and double fluorescent-labeled RNAs revealed moderate kinetic differenc es. Results with two systems, hammerhead ribozymes in I/II (small ribozyme, large substrate) and in I/III format (large ribozyme, small substrate), ar e reported.