期刊
RNA
卷 24, 期 1, 页码 114-124出版社
COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
DOI: 10.1261/rna.064014.117
关键词
glyoxal; RNA structure; in vivo RNA probing
资金
- National Science Foundation Plant Genome Research Program [NSF-IOS-1612170, NSF-IOS-1339282]
- Charles E. Kaufman Foundation of the Pittsburgh Foundation [KA2016-85222]
- Direct For Biological Sciences
- Division Of Integrative Organismal Systems [1612170] Funding Source: National Science Foundation
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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