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 32 P-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 magnesiumdependent 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.Hydroxyl radicals are important reactive species which have been widely used in studies of higher order nucleic acid structure and complexes formed between small molecules or proteins and nucleic acids (1, 2). Initial hydrogen abstraction from the ribose or the deoxyribose moiety is followed by a reaction cascade which results in cleavage of the phosphodiester backbone. Sequence-independent cleavage of both single-stranded and double-stranded DNA and RNA molecules can be analyzed at nucleotide resolution by denaturing polyacrylamide gel electrophoresis (PAGE) 1 of 32 P-labeled molecules. The formation of either higher order structure or specific complexes can lead to protection from such cleavage. A region of reduced cleavage, usually detected by denaturing PAGE, is referred to as a footprint. Footprinting experiments have typically been used to provide structural information on systems at equilibrium. When it is possible to measure footprints as a function of time, it then becomes possible to make inferences about rates and mechanisms of dynamic processes of which RNA folding is one example.

Exploiting graphical processing units for data‐parallel scientific applications

“…We used a derivative of N ‐hydroxypyridine‐2(1 H )‐thione that generates hydroxyl radicals upon photolysis to induce cleavage of r(CUG) exp . Previous studies have used N ‐hydroxypyridine‐2(1 H )‐thione to probe RNA structure in vitro 12. Thus, we synthesized 1‐hydroxy‐6‐thioxo‐1,6‐dihydropyridine‐2‐carboxylic acid (N‐HPT) to conjugate a hydroxyl‐radical‐producing warhead onto 2H‐4 using an amide bond (2H‐4‐HPT; Figure 2).…”

DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato

“…Among the sequences noteworthy here were RTL3 , poly(A) RNA polymerease , and histone methyltransferase , which the first two encode RNA processing enzymes and third one catalyzes histone methylation. Histone methylation and alternative RNA polyadenylation have been shown to be strongly correlated with light regulation of the gene expression in plants [ 35 – 38 ]. However, it’s not clear if these enzymes themselves could also be regulated by DNA methylation.…”

From equilibrium to kinetic footprinting of RNA structure

“…Recently, it has been shown that peroxynitrous acid is useful for analyzing events with half-lives of greater than ∼3 seconds. [19][20][21] The reagent is synthesized by oxidation of potassium peroxynitrite with peroxide followed by trapping with potassium hydroxide to yield the stable potassium salt. 22 Addition of potassium peroxynitrite to aqueous solutions buffered to pH 7 produces peroxynitrous acid which decomposes with a half-life of less than a second to generate hydroxyl radicals (Fig.…”