Glyoxals as in vivo RNA structural probes of guanine base-pairing.
Glyoxals as in vivo RNA structural probes of guanine base-pairing.
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DOI：
10.1261/rna.064014.117
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发表时间：
2018-01
期刊：
影响因子：
--
通讯作者：
Bevilacqua PC
中科院分区：
文献类型：
--
作者：
Mitchell D 3rd;Ritchey LE;Park H;Babitzke P;Assmann SM;Bevilacqua PC
阐明RNA在体内形成的折叠结构对于理解其功能至关重要。修饰未保护的核碱基的沃森-克里克（WC）面的化学试剂在结构解析中特别有用。硫酸二甲酯穿透细胞膜，并告知RNA碱基配对和二级结构，但仅修饰腺嘌呤和胞嘧啶的WC面。我们目前的乙二醛，甲基乙二醛，和苯乙二醛作为有效的体内试剂，靶向WC面对鸟嘌呤以及胞嘧啶和腺嘌呤。水稻（Oryza sativa）5.8S rRNA在体外通过逆转录和凝胶电泳读出的测试表明，几乎所有的鸟嘌呤在时间和pH值依赖性的方式进行特定的修改。随后在水稻，真核生物，枯草芽孢杆菌和大肠杆菌，革兰氏阳性菌和革兰氏阴性菌，分别体内试验表明，所有三种试剂进入活细胞没有事先膜透化或pH值调节周围的媒体和具体修改溶剂暴露的鸟嘌呤，胞嘧啶和腺嘌呤残基。
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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