In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
In vivo RNA structural probing of uracil and guanine base-pairing by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
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DOI：
10.1261/rna.067868.118
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发表时间：
2019-01
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;Renda AJ;Douds CA;Babitzke P;Assmann SM;Bevilacqua PC
rna的许多生物学功能源于其体内结构。同一RNA的结构在体外和体内可能不同，部分原因是受到从质子到次级代谢物再到蛋白质等分子的影响。修饰未受保护的RNA碱基的沃森-克里克（WC）表面的化学试剂报告了碱基配对的缺失，因此对确定RNA采用的结构具有价值。因此，人们一直在寻找能够报道活细胞中普遍存在的天然RNA结构的试剂。硫酸二甲酯（DMS）和乙二醛通过修饰腺嘌呤(A)、胞嘧啶(C)和鸟嘌呤(G)碱基，在体内穿透细胞膜并通知RNA二级结构。然而，尿嘧啶(U)碱基迄今尚未在体内进行鉴定。本文中，我们发现水溶性碳二亚胺(1-乙基-3-（3-二甲氨基丙基）碳二亚胺（EDC）能够在体内修饰U和G的WC面，有利于前者的核碱基约1.5倍，并且在真核生物水稻和革兰氏阴性细菌大肠杆菌中也是如此。EDC和乙二醛都以Gs为目标，EDC在典型的中性状态下与Gs反应，而乙二醛则需要Gs填充罕见的阴离子状态。因此，EDC可能更普遍有用；然而，通过比较EDC和乙二醛的反应性，可以在体内和全基因组范围内鉴定出具有扰动pka的Gs。总的来说，EDC与DMS的使用允许在体内探测所有四种RNA碱基的碱基配对状态。
Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson–Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli. While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.
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