A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry
A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry
复制标题
DOI：
10.1021/ja0704028
复制
发表时间：
2007-04-11
影响因子：
15
通讯作者：
Weeks, Kevin M.
中科院分区：
文献类型：
--
作者：
Mortimer, Stefanie A.;Weeks, Kevin M.
通过引物延伸(SHAPE)化学分析的选择性2‘-羟基酰化反应可以在任何RNA的单核苷酸分辨率下定量地评估局部核苷酸的灵活性。形状化学利用核糖2‘-羟基的亲核反应性的基于结构的门控，根据核苷酸的限制或柔性的程度。形状化学是用N-甲基异酸酐(NMIA)发展起来的，它只是中等亲电性，需要几十分钟才能形成核糖2‘-O-加合物。在这里，我们设计和评估了一种更有用的、快速有效的形状化学试剂。在反应羰基上引入硝基，生成1-甲基-7-硝基-7-硝基异酸酐(1M7)，得到一种试剂，该试剂与RNA反应更快，形成2‘-O-加合物，也更不稳定地进行有利的、自限的水解。使用1M7，在70名S中完成了对RNA结构的单核苷酸解析查询。用1M7进行的形状分析准确地报告了RNaseP特异域的二级和三级结构，并使该RNA的二级结构预测准确率高达91%。
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry allows local nucleotide flexibility to be quantitatively assessed at single nucleotide resolution in any RNA. SHAPE chemistry exploits structure-based gating of the nucleophilic reactivity of the ribose 2'-hydroxyl group by the extent to which a nucleotide is constrained or flexible. SHAPE chemistry was developed using N-methylisatoic anhydride (NMIA), which is only moderately electrophilic and requires tens of minutes to form ribose 2'-O-adducts. Here, we design and evaluate a significantly more useful, fast-acting, reagent for SHAPE chemistry. Introduction of a nitro group para to the reactive carbonyl to form 1-methyl-7-nitroisatoic anhydride (1M7) yields a reagent that both reacts significantly more rapidly with RNA to form 2'-O-adducts and is also more labile toward advantageous, self-limiting, hydrolysis. With 1M7, the single nucleotide resolution interrogation of the RNA structure is complete in 70 s. SHAPE analysis performed with 1M7 accurately reports the secondary and tertiary structure of the RNase P specificity domain and allows the secondary structure of this RNA to be predicted with up to 91% accuracy.