Poster abstracts

Poster number 90 submitted by David Mitchell III

Glyoxals as in vivo RNA structural probes of guanine base pairing

David Mitchell III (Department of Chemistry, Penn State University), Laura E. Ritchey (Department of Chemistry, Penn State University), Hongmarn Park (Department of Biochemistry and Molecular Biology, Penn State University), Paul Babitzke (Department of Biochemistry and Molecular Biology, Penn State University), Sarah M. Assmann (Department of Biology, Penn State University), Philip C. Bevilacqua (Department of Chemistry, Department of Biochemistry and Molecular Biology, Penn State University)

Abstract:
Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice (Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli, Gram positive and Gram negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues.

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Keywords: Glyoxal, RNA structure, in vivo RNA probing