Franceschi José A Reyes, Cárdenas Emilio L, Klein Brandon J C, Weidmann Chase A, Garner Amanda L
Program in Chemical Biology, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109, United States.
bioRxiv. 2025 Jul 21:2025.07.21.665968. doi: 10.1101/2025.07.21.665968.
Interrogating RNA-small molecule interactions inside cells is critical for advancing RNA-targeted drug discovery. In particular, chemical probing technologies that both identify small molecule-bound RNAs and define their binding sites in the complex cellular environment will be key for establishing the on-target activity necessary for successful hit-to-lead campaigns. Using the small molecule metabolite preQ and its cognate riboswitch RNA as a model, herein we describe a chemical probing strategy for filling this technological gap. Building on well-established RNA acylation chemistry employed by click selective 2'-hydroxyl acylation analyzed by primer extension (icSHAPE) probes, we developed an icSHAPE-based preQ probe that retains biological activity in a preQ riboswitch reporter assay and successfully enriches the preQ riboswitch from living bacterial cells. Further, we map the preQ binding site on probe-modified riboswitch RNA by mutational profiling (MaP). As the need for rapid profiling of on- and off-target small molecule interactions continues to grow, this chemical probing strategy offers a method to interrogate cellular RNA-small molecule interactions and support the future development of RNA-targeted therapeutics.
探究细胞内RNA与小分子的相互作用对于推进以RNA为靶点的药物研发至关重要。特别是，能够在复杂的细胞环境中识别与小分子结合的RNA并确定其结合位点的化学探测技术，将是成功开展从苗头化合物到先导化合物转化研究所需的靶向活性确立的关键。以小分子代谢物preQ及其同源核糖开关RNA为模型，我们在此描述一种化学探测策略来填补这一技术空白。基于通过引物延伸分析的点击选择性2'-羟基酰化（icSHAPE）探针所采用的成熟RNA酰化化学，我们开发了一种基于icSHAPE的preQ探针，该探针在preQ核糖开关报告基因检测中保留生物活性，并成功从活细菌细胞中富集preQ核糖开关。此外，我们通过突变谱分析（MaP）绘制了探针修饰的核糖开关RNA上的preQ结合位点。随着对快速分析靶向和非靶向小分子相互作用的需求持续增长，这种化学探测策略提供了一种探究细胞内RNA与小分子相互作用的方法，并支持以RNA为靶点的治疗药物的未来发展。
