Bizebard Thierry, Arluison Véronique, Bockelmann Ulrich
Expression Génétique Microbienne, UMR8261 CNRS/Université Paris 7, IBPC, Paris, France.
Université Paris Diderot & LLB/CEA/CNRS, Gif-sur-Yvette, France.
Methods Mol Biol. 2018;1737:301-319. doi: 10.1007/978-1-4939-7634-8_17.
In recent years, single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique to study macromolecular interactions. The chief advantages of smFRET analysis compared to bulk measurements include the possibility to detect sample heterogeneities within a large population of molecules and the facility to measure kinetics without needing the synchronization of intermediate states. As such, the methodology is particularly well adapted to observe and analyze RNA/RNA and RNA/protein interactions involved in small noncoding RNA-mediated gene regulation networks. In this chapter, we describe and discuss protocols that can be used to measure the dynamics of these interactions, with a particular emphasis on the advantages-and experimental pitfalls-of using the smFRET methodology to study sRNA-based biological systems.
近年来，单分子荧光共振能量转移（smFRET）已成为研究大分子相互作用的一种强大技术。与整体测量相比，smFRET分析的主要优势包括能够检测大量分子中的样品异质性，以及无需中间状态同步即可测量动力学的便利性。因此，该方法特别适合观察和分析参与小非编码RNA介导的基因调控网络的RNA/RNA和RNA/蛋白质相互作用。在本章中，我们描述并讨论可用于测量这些相互作用动力学的方案，特别强调使用smFRET方法研究基于sRNA的生物系统的优势和实验陷阱。
