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RNA. 2018 Jan;24(1):114-124. doi: 10.1261/rna.064014.117. Epub 2017 Oct 13.

Glyoxals as in vivo RNA structural probes of guanine base-pairing.

Author information

1
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
2
Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
3
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
4
Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

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.

KEYWORDS:

RNA structure; glyoxal; in vivo RNA probing

PMID:
29030489
PMCID:
PMC5733565
[Available on 2019-01-01]
DOI:
10.1261/rna.064014.117
[Indexed for MEDLINE]

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