Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, United States.
Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, United States; McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, United States.
Methods. 2020 Nov 1;183:76-83. doi: 10.1016/j.ymeth.2020.01.015. Epub 2020 Jan 25.
RNA regulation is influenced by the dynamic changes in conformational accessibility on the transcript. Here we discuss the initial validation of a cell-free antisense probing method for structured RNAs, using the Tetrahymena group I intron as a control target. We observe changes in signal that qualitatively match prior traditional DMS footprinting experiments. Importantly, we have shown that application of this technique can elucidate new RNA information given its sensitivity for detecting rare intermediates that are not as readily observed by single-hit kinetics chemical probing techniques. Observing changes in RNA accessibility has broad applications in determining the effect that regulatory elements have on regional structures. We speculate that this method could be useful in quickly observing those interactions, along with other phenomena that influence RNA accessibility including RNA-RNA interactions and small molecules.
RNA 的调控受到转录本构象可及性动态变化的影响。在这里，我们讨论了一种用于结构 RNA 的无细胞反义探测方法的初步验证，使用四膜虫组 I 内含子作为对照靶标。我们观察到信号的变化与先前的传统 DMS 足迹实验定性匹配。重要的是，我们已经表明，该技术的应用可以阐明新的 RNA 信息，因为它具有检测稀有中间体的敏感性，而这些中间体不容易被单次命中动力学化学探测技术观察到。观察 RNA 可及性的变化在确定调节元件对区域结构的影响方面具有广泛的应用。我们推测，该方法可能有助于快速观察这些相互作用，以及影响 RNA 可及性的其他现象，包括 RNA-RNA 相互作用和小分子。
