Time-resolved hydroxyl-radical footprinting of RNA using Fe(II)-EDTA

Chemical footprinting methods have been used extensively to probe the struc tures of biologically important RNAs at nucleotide resolution. One of these methods, hydroxyl-radical footprinting, has recently been employed to stud y the kinetics of RNA folding. Hydroxyl radicals can be generated by a numb er of different methods, including Fe(ll)-EDTA complexes, synchrotron radia tion, and peroxynitrous acid disproportionation. The latter two methods hav e been used for kinetic studies of RNA folding. We have taken advantage of rapid hydroxyl-radical generation by Fe(II)-EDTA-hydrogen peroxide solution s 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 footprin ting on the hairpin ribozyme, a small endoribonuclease and RNA ligase. (C) 2001 Academic Press.