Fluorescence Resonance Energy Transfer Analysis of RNase L-Catalyzed Oligonucleotide Cleavage

Abstract

A method is described for monitoring the cleavage of an oligoribonucleotide substrate by the 2-5A-dependent RNase L based on fluorescence resonance energy transfer (FRET). The oligoribonucleotide, rC₁₁U₂C₇, was labeled covalently at its 5‵-terminus with fluorescein and at its 3‵-terminus with rhodamine to provide a substrate for RNase L. On cleavage, the fluorescence at 538 nm (with 485 nm excitation) increased by a factor of 2.8, allowing real-time quantitation of the reaction progress. The method was performed easily in a 96-well plate format and allowed quantitative high throughput analyses of RNase L activity with different activators.