RNA structure investigation: a deuterium kinetic isotope effect/hydroxyl radical cleavage experiment
Ingle, Shakti Singh
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The hydroxyl radical is widely used as a high-resolution footprinting agent for DNA and RNA. The hydroxyl radical abstracts a hydrogen atom from the sugar- phosphate backbone of a nucleic acid molecule, creating a sugar-based radical that eventually results in a strand break. It was shown previously that replacement of deoxyribose hydrogen atoms with deuterium results in a kinetic isotope effect (KIE) on hydroxyl radical cleavage of DNA. The KIE correlates well with the solvent accessible surface area of a deoxyribose hydrogen atom in DNA. We chose the structurally well-defmed sarcin-ricin loop (SRL) RNA molecule as a model system to extend the deuterium KIE/hydroxyl radical cleavage experiment to RNA. We observed a substantial KIE upon deuteration of the 5'-carbon of the ribose. Values ranged from 1.20 to 1.96, and depended on the position of the residue within the SRL. We found a smaller KIE upon 4'-deuteration. Values ranged from 1.05 to 1.23. Values of 5' and 4' KIEs correlate with the extent of cleavage and with the solvent accessible surface areas of ribose hydrogen atoms ofthe SRL. Gel electrophoresis of cleavage products reveals that the strand break is terminated at the 5' end by multiple chemical species. Upon 3'-radiolabeling a specifically 5'-deuterated SRL RNA molecule, we observed a KIE on the production of a cleavage product having a gel mobility different from that of a phosphate-terminated RNA strand. Reduction with sodium borohydride gave rise to an RNA fragment terminated by a 5'-hydroxyl group. These experiments are consistent with 5' hydrogen abstraction by the hydroxyl radical producing a 5'-aldehyde-terminated RNA strand that retains the nucleotide from which the hydrogen atom was abstracted. This is the first report of such a species. This chemistry has important implications for the interpretation of structural analysis experiments on RNA that rely on primer extension to synthesize eDNA copies of hydroxyl radical cleavage products. The different 5'-terminated products resulting from hydroxyl radical cleavage at a given nucleotide would yield cDNAs of two different lengths, thereby distributing the cleavage intensity over two nucleotides instead ofone and lowering the resolution ofthe experiment.
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