Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
J Am Chem Soc. 2012 Aug 15;134(32):13160-3. doi: 10.1021/ja304027m. Epub 2012 Aug 1.
Many RNA structures are composed of simple secondary structure elements linked by a few critical tertiary interactions. SHAPE chemistry has made interrogation of RNA dynamics at single-nucleotide resolution straightforward. However, de novo identification of nucleotides involved in tertiary interactions remains a challenge. Here we show that nucleotides that form noncanonical or tertiary contacts can be detected by comparing information obtained using two SHAPE reagents, N-methylisatoic anhydride (NMIA) and 1-methyl-6-nitroisatoic anhydride (1M6). Nucleotides that react preferentially with NMIA exhibit slow local nucleotide dynamics and usually adopt the less common C2'-endo ribose conformation. Experiments and first-principles calculations show that 1M6 reacts preferentially with nucleotides in which one face of the nucleobase allows an unhindered stacking interaction with the reagent. Differential SHAPE reactivities were used to detect noncanonical and tertiary interactions in four RNAs with diverse structures and to identify preformed noncanonical interactions in partially folded RNAs. Differential SHAPE reactivity analysis will enable experimentally concise, large-scale identification of tertiary structure elements and ligand binding sites in complex RNAs and in diverse biological environments.
许多 RNA 结构由通过少数关键三级相互作用连接的简单二级结构元件组成。SHAPE 化学使在单核苷酸分辨率下对 RNA 动力学的询问变得简单直接。然而，涉及三级相互作用的核苷酸的从头鉴定仍然是一个挑战。在这里，我们表明，通过比较使用两种 SHAPE 试剂（N-甲基异邻苯二甲酰亚胺（NMIA）和 1-甲基-6-硝基异邻苯二甲酰亚胺（1M6）获得的信息，可以检测到形成非规范或三级接触的核苷酸。优先与 NMIA 反应的核苷酸表现出缓慢的局部核苷酸动力学，并且通常采用不太常见的 C2'-endo 核糖构象。实验和第一性原理计算表明，1M6 优先与允许试剂与碱基的一个面进行无阻碍堆积相互作用的核苷酸反应。差异 SHAPE 反应性被用于检测具有不同结构的四种 RNA 中的非规范和三级相互作用，并鉴定部分折叠的 RNA 中的预形成非规范相互作用。差异 SHAPE 反应性分析将能够在复杂的 RNA 中和在不同的生物环境中，通过实验简洁地大规模鉴定三级结构元件和配体结合位点。
