Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Methods. 2019 Jun 1;162-163:3-11. doi: 10.1016/j.ymeth.2019.04.002. Epub 2019 Apr 3.
RNA structures and their dynamic fluctuations lie at the heart of understanding key biological process such as transcription, splicing, translation and RNA decay. While conventional bulk assays have proven to identify and characterize key pathway intermediates, the generally dynamic nature of RNA structures renders the information obtained from time and ensemble averaging techniques necessarily lacking in critical details. Here we detail Single-Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS), a method that readily monitors structural fluctuations of single RNA molecules through the repetitive interaction of fluorescent probes with an unlabeled, surface-immobilized RNA target of virtually any length and in any biological context. In addition, we demonstrate the broad applicability of SiM-KARTS by kinetically fingerprinting the binding of cognate tRNA ligand to single immobilized T-box riboswitch molecules. SiM-KARTS represents a valuable tool for probing biologically relevant structure and interaction features of potentially many diverse RNA metabolic pathways.
RNA 结构及其动态变化是理解转录、剪接、翻译和 RNA 降解等关键生物学过程的核心。虽然传统的批量分析已被证明可以识别和表征关键途径中间产物，但 RNA 结构的普遍动态性质使得从时间和整体平均技术获得的信息必然缺乏关键细节。在这里，我们详细介绍了 RNA 瞬态结构的单分子动力学分析 (SiM-KARTS)，这是一种通过荧光探针与未标记的、表面固定的 RNA 靶标反复相互作用，轻松监测单个 RNA 分子结构波动的方法，该方法几乎可以在任何长度和任何生物环境下监测任何 RNA 靶标。此外，我们通过动态鉴定单个固定 T 盒核酶分子上同源 tRNA 配体的结合，证明了 SiM-KARTS 的广泛适用性。SiM-KARTS 代表了一种有价值的工具，可用于探测潜在的许多不同 RNA 代谢途径中与生物学相关的结构和相互作用特征。
