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

N-Hydroxypyridine-2(1H)-thione (N-HPT) has been studied as a photochemical source of hydroxyl radicals far use in photoinitiated nucleic acid footprin ting experiments. Steady-state photolysis of dilute aqueous solutions of N- HPT at 350 nm in the presence of a 385 nucleotide P-32-labeled RNA, the Tet rahymena L-21 ribozyme, resulted in cleavage of the RNA at nucleotide resol ution. No cleavage of the RNA occurred in the absence of light or in the ab sence of N-HPT. Photolysis of the analogous pyridine lacking the N-hydroxyl group did not result in detectable amounts of RNA cleavage. The addition o f RNA to preirradiated solutions of N-HPT gave no apparent RNA cleavage pro ducts, suggesting that the photoproducts of N-HPT do not result in RNA modi fication. Cleavage of RNA, upon photolysis in the presence of N-HPT, occurr ed in a sequence-independent fashion with double-stranded RNA being cleaved as efficiently as single-stranded RNA. Based on these observations, we con clude that photochemically generated diffusable hydroxyl radicals an respon sible for the RNA cleavage. Experiments involving the photolysis of N-HPT i n the presence of the Tetrahymena ribozyme and magnesium showed a magnesium -dependent protection from RNA cleavage due to formation of a folded RNA te rtiary structure. The locations and amount of protection were identical to those observed in footprinting experiments performed with other hydroxyl ra dical sources. The presence of N-HPT had no effect on either the rate of fo lding or the catalytic activity of the folded RNA, indicating that this rea gent does not disrupt RNA tertiary structure or otherwise affect activity. Thus, N-HPT is established as a new reagent for use in photoinitiated RNA f ootprinting experiments.