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PMID: 29030489 Published · ppublish English Journal Article

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

RNA (New York, N.Y.) ·Vol. 24 ·No. 1 ·2018-00-00 ·Pages 114-124

Mitchell D, Ritchey LE, Park H, Babitzke P, Assmann SM, Bevilacqua PC

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
MeSH Terms
Bacillus subtilis Base Pairing Escherichia coli Glyoxal/chemistry Guanine/chemistry,metabolism Oryza RNA Probes/chemistry,metabolism RNA, Bacterial/chemistry,metabolism RNA, Plant/chemistry,metabolism Staining and Labeling
Chemicals
RNA Probes RNA, Bacterial RNA, Plant Glyoxal Guanine
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Mitchell David ORCID
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. | Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Ritchey Laura E
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. | Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Park Hongmarn
Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. | Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Babitzke Paul
Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. | Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Assmann Sarah M
Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Bevilacqua Philip C ORCID
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. | Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. | Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1469-9001
DOI
10.1261/rna.064014.117
Published
2018-00-00
Epub
2017-00-13
Pages
114-124
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC5733565
Subset
IM
External Links
PubMed source DOI: 10.1261/rna.064014.117 PMC Full Text View journal details
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