Department of Chemistry & Biochemistry, University of the Sciences, Philadelphia, PA 19104 United States.
Department of Chemistry & Biochemistry, University of the Sciences, Philadelphia, PA 19104 United States.
Methods. 2019 Sep 1;167:54-65. doi: 10.1016/j.ymeth.2019.05.017. Epub 2019 May 23.
Interrogating non-coding RNA structures and functions with small molecules is an area of rapidly increasing interest among biochemists and chemical biologists. However, many biochemical approaches to monitoring RNA structures are time-consuming and low-throughput, and thereby are only of limited utility for RNA-small molecule studies. Fluorescence-based techniques are powerful tools for rapid investigation of RNA conformations, dynamics, and interactions with small molecules. Many fluorescence methods are amenable to high-throughput analysis, enabling library screening for small molecule binders. In this review, we summarize numerous fluorescence-based approaches for identifying and characterizing RNA-small molecule interactions. We describe in detail a high-information content dual-reporter FRET assay we developed to characterize small molecule-induced conformational and stability changes. Our assay is uniquely suited as a platform for both small molecule discovery and thorough characterization of RNA-small molecule binding mechanisms. Given the growing recognition of non-coding RNAs as attractive targets for therapeutic intervention, we anticipate our FRET assay and other fluorescence-based techniques will be indispensable for the development of potent and specific small molecule inhibitors targeting RNA.
小分子与非编码 RNA 结构和功能的相互作用是生物化学家和化学生物学家日益关注的一个领域。然而，许多用于监测 RNA 结构的生化方法既耗时又低通量，因此对于 RNA-小分子研究的应用具有很大的局限性。基于荧光的技术是快速研究 RNA 构象、动态和与小分子相互作用的有力工具。许多荧光方法适用于高通量分析，可用于小分子结合物文库的筛选。在这篇综述中，我们总结了许多基于荧光的方法来鉴定和表征 RNA-小分子相互作用。我们详细描述了我们开发的一种高信息量的双报告 FRET 测定法，用于表征小分子诱导的构象和稳定性变化。我们的测定法特别适合作为小分子发现和 RNA-小分子结合机制的全面表征的平台。鉴于非编码 RNA 作为治疗干预的有吸引力的靶标越来越受到认可，我们预计我们的 FRET 测定法和其他基于荧光的技术对于开发针对 RNA 的有效和特异性小分子抑制剂将是不可或缺的。
