Nature of guanine oxidation in RNA via the flash-quench technique versus direct oxidation by a metal oxo complex.

Abstract

Oxidation of RNA can be effected by two different techniques: a photochemical, electron-transfer method termed "flash-quench" and direct oxidation by metal oxo complexes. The flash-quench method produces selective oxidation using a metal photosensitizer, tris(bipyridyl)ruthenium(III) trichloride (Ru(bpy)(3)(3+)), and quencher, pentaamminechlorocobalt(III) chloride (Co(NH(3))(5)Cl(2+)). We have optimized the flash-quench technique for the following RNAs: tRNA(Phe), human ferritin iron-responsive element (IRE), and a mutated human ferritin IRE. We have also employed a chemical footprinting technique involving the oxoruthenium(IV) complex (Ru(tpy)(bpy)O(2+) (tpy = 2,2',2''-terpyridine; bpy = 2,2'-bipyridine)) to oxidize guanine. Comparison of the two methods shows that the flash-quench technique provides a visualization of nucleotide accessibility for a static conformation of RNA while the Ru(tpy)(bpy)O(2+) complex selectively oxidizes labile guanines and gives a visualization of a composite of multiple conformations of the RNA structure.