Chan Dalen, Feng Chao, Zhen Yuran, Flynn Ryan A, Spitale Robert C
Department of Pharmaceutical Sciences, University of California, Irvine , Irvine, California 92697, United States.
Department of Chemistry, Stanford University , Stanford, California 94305, United States.
Biochemistry. 2017 Apr 4;56(13):1811-1814. doi: 10.1021/acs.biochem.7b00128. Epub 2017 Mar 21.
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.
RNA分子依赖于对细胞功能至关重要的结构元件。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其效用至关重要。选择性羟基酰化已成为分析RNA结构的常用方法。然后可以通过逆转录或突变酶谱分析来鉴定2'-羟基酰化产生的酯产物。通过引物延伸（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这一事实。尽管其很重要，但尚未对SHAPE亲电试剂形成稳定RNA加合物的能力进行直接比较。在此，我们进行了水解稳定性实验的系统分析，以证明糠酰咪唑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，即使在活细胞中，用二硫苏糖醇淬灭也可以控制与糠酰酰亚胺唑SHAPE试剂的酰化反应。这些结果对于我们理解SHAPE实验的生化细节很重要。
