Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA.
Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) for direct analysis of covalent adducts and of nucleotide flexibility in RNA.
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DOI：
10.1038/nprot.2011.373
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发表时间：
2011-10-06
期刊：
影响因子：
14.8
通讯作者：
Weeks KM
中科院分区：
文献类型：
--
作者：
Steen KA;Siegfried NA;Weeks KM
RNA SHAPE化学基于构象柔性核苷酸的2′-羟基与亲电子SHAPE试剂的反应产生定量的单核苷酸解析结构信息。然而，SHAPE技术受到通过引物延伸检测RNA修饰位点的要求的限制。引物延伸导致RNA的5′和3′末端的信息丢失，需要多个实验步骤。在这里，我们描述了RNA酶检测的SHAPE（通过保护免受核糖核酸外切酶分析的选择性2′-羟基酰化），其使用进行性的3′→5′核糖核酸外切酶，RNA酶R，在单管实验中检测5′端标记RNA中的共价加合物。RNase R降解RNA，但在核糖2′-羟基或核碱基的配对面上分别定量终止含有共价加合物的核苷酸的3′端的三个和四个核苷酸。我们通过描述配体诱导的E.大肠杆菌硫胺素焦磷酸核糖开关RNA。RNA酶检测SHAPE是一种简便的两天方法，可用于分析任何RNA分子中的各种共价加合物，包括不适合通过引物延伸分析的短RNA和在其5′或3′末端具有重要功能结构的RNA。
RNA SHAPE chemistry yields quantitative, single nucleotide resolution structural information based on the reaction of the 2′-hydroxyl group of conformationally flexible nucleotides with electrophilic SHAPE reagents. However, SHAPE technology has been limited by the requirement that sites of RNA modification be detected by primer extension. Primer extension results in loss of information at both the 5′ and 3′ ends of an RNA and requires multiple experimental steps. Here we describe RNase-detected SHAPE (Selective 2′-Hydroxyl Acylation analyzed by Protection from Exoribonuclease) that uses a processive, 3′→5′ exoribonuclease, RNase R, to detect covalent adducts in 5′-end labeled RNA in a one-tube experiment. RNase R degrades RNA but stops quantitatively three and four nucleotides 3′ of a nucleotide containing a covalent adduct at the ribose 2′-hydroxyl or the pairing face of a nucleobase, respectively. We illustrate this technology by characterizing ligand-induced folding for the E. coli thiamine pyrophosphate riboswitch RNA. RNase-detected SHAPE is a facile, two-day approach that can be used to analyze diverse covalent adducts in any RNA molecule, including short RNAs not amenable to analysis by primer extension and RNAs with functionally important structures at their 5′ or 3′ ends.