Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
J Am Chem Soc. 2010 Jul 28;132(29):9940-3. doi: 10.1021/ja103781u.
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful approach for characterizing RNA structure and dynamics at single-nucleotide resolution. However, SHAPE technology is limited, sometimes severely, because primer extension detection obscures structural information for approximately 15 nts at the 5' end and 40-60 nts at the 3' end of the RNA. Moreover, detection by primer extension is more complex than the actual structure-selective chemical interrogation step. Here we quantify covalent adducts in RNA directly by adduct-inhibited exoribonuclease degradation. RNA 2'-O-adducts block processivity of a 3'-->5' exoribonuclease, RNase R, to produce fragments that terminate three nucleotides 3' of the modification site. We analyzed the structure of the native thiamine pyrophosphate (TPP) riboswitch aptamer domain and identified large changes in local nucleotide dynamics and global RNA structure upon ligand binding. In addition to numerous changes that can be attributed to ligand recognition, we identify a single nucleotide bulge register shift, distant from the binding site, that stabilizes the ligand-bound structure. Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) should prove broadly useful for facile structural analysis of small noncoding RNAs and for RNAs that have functionally critical structures at their 5' and 3' ends.
通过引物延伸分析的选择性 2'-羟基酰化(SHAPE)是一种在单核苷酸分辨率下表征 RNA 结构和动态的强大方法。然而,SHAPE 技术受到限制,有时限制非常严重,因为引物延伸检测会掩盖 RNA 5'端约 15 个核苷酸和 3'端约 40-60 个核苷酸的结构信息。此外,引物延伸检测比实际的结构选择性化学询问步骤更复杂。在这里,我们通过加合物抑制外切核酸酶降解直接定量 RNA 中的共价加合物。RNA 2'-O 加合物阻止 3'-->5'外切核酸酶 RNase R 的连续性,产生终止于修饰部位三个核苷酸 3'的片段。我们分析了天然硫胺素焦磷酸(TPP)核糖开关适体结构域的结构,并在配体结合时鉴定了局部核苷酸动态和全局 RNA 结构的大量变化。除了可以归因于配体识别的许多变化外,我们还确定了一个远离结合位点的单个核苷酸凸起记录移位,该移位稳定了配体结合结构。通过外切核酸酶保护分析的选择性 2'-羟基酰化(RNase 检测的 SHAPE)应该被证明对小非编码 RNA 的简单结构分析以及在其 5'和 3'末端具有功能关键结构的 RNA 非常有用。