Probing RNA structures with hydroxyl radicals

Fe(II)-EDTA can be used to conveniently generate hydroxyl radicals to promote cleavage of RNA at nucleotide resolution. Two procedures are described, involving the generation of free radicals from solvated molecular oxygen and from hydrogen peroxide added to the RNA solution. Unlike other footprinting reagents, hydroxyl radicals cleave the sugar-phosphate backbone at every residue and thus provide uniform cleavage in a given RNA secondary structure. Because some positions become protected by tertiary folding, this reagent is useful for monitoring the global folding of RNA at equilibrium.