Studies of RNA cleavage by photolysis of N-hydroxypyridine-2(1H)-thione. A new photochemical footprinting method

Abstract

N-Hydroxypyridine-2(1H)-thione (N-HPT) has been studied as a photochemical source of hydroxyl radicals for use in photoinitiated nucleic acid footprinting experiments. Steady-state photolysis of dilute aqueous solutions of N-HPT at 350 nm in the presence of a 385 nucleotide 32P-labeled RNA, the Tetrahymena L-21 ribozyme, resulted in cleavage of the RNA at nucleotide resolution. No cleavage of the RNA occurred in the absence of light or in the absence of N-HPT. Photolysis of the analogous pyridine lacking the N-hydroxyl group did not result in detectable amounts of RNA cleavage. The addition of RNA to preirradiated solutions of N-HPT gave no apparent RNA cleavage products, suggesting that the photoproducts of N-HPT do not result in RNA modification. Cleavage of RNA, upon photolysis in the presence of N-HPT, occurred in a sequence-independent fashion with double-stranded RNA being cleaved as efficiently as single-stranded RNA. Based on these observations, we conclude that photochemically generated diffusable hydroxyl radicals are responsible for the RNA cleavage. Experiments involving the photolysis of N-HPT in the presence of the Tetrahymena ribozyme and magnesium showed a magnesium-dependent protection from RNA cleavage due to formation of a folded RNA tertiary structure. The locations and amount of protection were identical to those observed in footprinting experiments performed with other hydroxyl radical sources. The presence of N-HPT had no effect on either the rate of folding or the catalytic activity of the folded RNA, indicating that this reagent does not disrupt RNA tertiary structure or otherwise affect activity. Thus, N-HPT is established as a new reagent for use in photoinitiated RNA footprinting experiments.