Chemical probing of RNA with the hydroxyl radical at single-atom resolution.
Bottom Line: We conclude that hydroxyl radical abstracts a 5'-hydrogen atom, leading to RNA strand cleavage.We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA.Cleavage at U exhibits a large 5' deuterium kinetic isotope effect, a potential signature of a base triple.
Affiliation: Department of Chemistry, Boston University, Boston, MA 02215, USA.Show MeSH
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Mentions: The hydroxyl radical produces a strand break in a nucleic acid molecule by abstracting a hydrogen atom from the sugar-phosphate backbone (11). There are hydrogen atoms attached to each of the five ribose carbons (Figure 1A), so in principle the hydroxyl radical could abstract any of these hydrogens. To interpret the hydroxyl radical cleavage experiment, it is presumed that the solvent accessibility of a hydrogen atom modulates its reactivity (8,9,12), thereby yielding a map of solvent accessible surface area that is based on the relative extent of cleavage at each nucleotide of an RNA. So, while the structural resolution of the experiment is conventionally defined by how often a nucleotide is cleaved, the chemistry underlying the method actually interrogates the accessibilities of single hydrogen atoms (11,13). If we could weigh the contributions of individual hydrogen atoms to the cleavage pattern, the resolution of this chemical probe experiment would reach the single-atom level.
Affiliation: Department of Chemistry, Boston University, Boston, MA 02215, USA.