单分子 FRET 研究 RNA 结构重排和 RNA-RNA 相互作用。
Single-Molecule FRET Studies of RNA Structural Rearrangements and RNA-RNA Interactions.
机构信息
T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA.
Department of Molecular Biology and Genetics, Johns Hopkins Medical School, Baltimore, MD, USA.
出版信息
Methods Mol Biol. 2022;2518:271-289. doi: 10.1007/978-1-0716-2421-0_16.
RNA-guided regulation of gene expression is found in all cell types. In this mode of regulation, antisense interactions between the regulatory RNA and its target are typically facilitated by a protein partner. Single-molecule fluorescence microscopy is a powerful tool for dissecting the conformational states and intermediates that contribute to target recognition. This chapter describes protocols for studying target recognition by bacterial small RNAs and their chaperone Hfq on the single-molecule level, using a total internal reflection fluorescence microscope. The sections cover the design of suitable RNA substrates for sRNA-mRNA annealing reactions, preparation of internally labeled mRNA for detecting conformational changes in the target, and key steps of the data analysis. These protocols can be adapted to other RNA-binding proteins that chaperone RNA interactions.
RNA 指导的基因表达调控存在于所有细胞类型中。在这种调节模式中，调控 RNA 与其靶标之间的反义相互作用通常由蛋白质伴侣来促进。单分子荧光显微镜是一种强大的工具，可用于剖析有助于靶标识别的构象状态和中间产物。本章描述了使用全内反射荧光显微镜在单分子水平上研究细菌小 RNA 及其伴侣蛋白 Hfq 对靶标的识别的方案。这些章节涵盖了设计适用于 sRNA-mRNA 退火反应的 RNA 底物、为检测靶标中构象变化制备内部标记的 mRNA 以及数据分析的关键步骤。这些方案可以适应其他具有 RNA 相互作用伴侣功能的 RNA 结合蛋白。
Zotero 插件