Time-Resolved Hydroxyl-Radical Footprinting of RNA Using Fe(II)-EDTA

Abstract

Chemical footprinting methods have been used extensively to probe the structures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to study the kinetics of RNA folding. Hydroxyl radicals can be generated by a number of different methods, including Fe(II)-EDTA complexes, synchrotron radiation, and peroxynitrous acid disproportionation. The latter two methods have been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solutions to develop a benchtop method to study folding kinetics of RNA complexes. This technique can be performed using commercially available chemicals, and can be used to accurately define RNA folding rate constants slower than 6 min−1. Here we report the method and an example of time-resolved footprinting on the hairpin ribozyme, a small endoribonuclease and RNA ligase.