Moon Michelle H, Hilimire Thomas A, Sanders Allix M, Schneekloth John S
Chemical Biology Laboratory , National Cancer Institute , Frederick , Maryland 21702 , United States.
Biochemistry. 2018 Aug 7;57(31):4638-4643. doi: 10.1021/acs.biochem.7b01141. Epub 2018 Jan 31.
In recent years, there has been dramatic growth in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA-small molecule interactions. While a host of techniques exist to measure RNA-small molecule interactions, many of these have drawbacks that make them intractable for routine use and are often not broadly applicable. A newer technology called microscale thermophoresis (MST), which measures the directed migration of a molecule and/or molecule-ligand complex along a temperature gradient, can be used to measure binding affinities using very small amounts of sample. The high sensitivity of this technique enables measurement of affinity constants in the nanomolar and micromolar range. Here, we demonstrate how MST can be used to study a range of biologically relevant RNA interactions, including peptide-RNA interactions, RNA-small molecule interactions, and displacement of an RNA-bound peptide by a small molecule.
近年来，RNA研究取得了显著进展。RNA已从被认为是分子生物学中心法则中的一个中间体，转变为一种具有多种结构和功能的分子，参与生物学的各个方面。随着新功能的迅速发现，很明显需要RNA靶向小分子探针来研究RNA生物学，并阐明基于RNA-小分子相互作用的治疗潜力。虽然存在许多测量RNA-小分子相互作用的技术，但其中许多技术都有缺点，使其难以常规使用，而且通常不具有广泛适用性。一种名为微尺度热泳（MST）的较新技术，它测量分子和/或分子-配体复合物沿温度梯度的定向迁移，可用于使用极少量样品测量结合亲和力。该技术的高灵敏度能够测量纳摩尔和微摩尔范围内的亲和常数。在这里，我们展示了MST如何用于研究一系列生物学相关的RNA相互作用，包括肽-RNA相互作用、RNA-小分子相互作用以及小分子取代RNA结合肽。
