Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts
Comparative Analysis Reveals Furoyl in Vivo Selective Hydroxyl Acylation Analyzed by Primer Extension Reagents Form Stable Ribosyl Ester Adducts
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DOI：
10.1021/acs.biochem.7b00128
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发表时间：
2017-04-04
期刊：
影响因子：
2.9
通讯作者：
Spitale, Robert C.
中科院分区：
文献类型：
--
作者：
Chan, Dalen;Feng, Chao;Spitale, Robert C.
RNA分子依赖于对细胞功能至关重要的结构元素。探测RNA结构的化学方法已成为表征RNA功能的必要组成部分。因此，了解这些方法的局限性和特性对于它们的使用是至关重要的。选择性羟基酰化已成为分析RNA结构的常用方法。酯产物作为2'-羟基酰化的结果，可以通过逆转录或突变酶分析来鉴定。引物延伸（SHAPE）实验分析的选择性羟基酰化的核心方面是在2'-羟基上形成稳定的酯加合物：尽管它很重要，但SHAPE亲电试剂在制造稳定的RNA加合物的能力方面还没有直接的比较。在此，我们对水解稳定性实验进行了系统的分析，证明了呋喃咪唑SHAPE试剂即使在高温下也能形成稳定的酯加合物。我们还证明了与呋喃酰基咪唑SHAPE试剂的酰化反应可以通过二硫苏糖醇猝灭来控制，甚至在活细胞中也是如此。这些结果对于我们理解SHAPE实验的生化细节非常重要。
RNA molecules depend on structural elements that are critical for cellular function. Chemical methods for probing RNA structure have emerged as a necessary component of characterizing RNA function. As such, understanding the limitations and idiosyncrasies of these methods is essential for their utility. Selective hydroxyl acylation has emerged as a common method for analyzing RNA structure. Ester products as a result of 2'-hydroxyl acylation can then,be identified through reverse transcription or mutational enzyme profiling. The central aspect of selective hydroxyl acylation analyzed by primer extension (SHAPE) experiments is the fact that stable ester adducts are formed on the 2'-hydroxyl: Despite its importance, there has not been a direct comparison of SHAPE electrophiles for their ability to make stable RNA adducts. Herein, we conduct a systematic analysis of hydrolysis stability experiments to demonstrate that furoyl imidazole SHAPE reagents form stable ester adducts even at elevated temperatures. We also demonstrate that the acylation reaction with the furoyl acylimidaole SHAPE reagent can be controlled with dithiothreitol quenching,, even in live cells. These results are important for our understanding of the biochemical details of the SHAPE experiment.