Zafferani Martina, Muralidharan Dhanasheel, Montalvan Nadeska I, Hargrove Amanda E
Department of Chemistry, Duke University 124 Science Drive Durham NC 27705 USA
RSC Chem Biol. 2022 Jun 6;3(7):905-915. doi: 10.1039/d2cb00015f. eCollection 2022 Jul 6.
The exponential increase in the discovery and characterization of RNA tertiary structures has highlighted their active role in a variety of human diseases, yet often their interactome and specific function remain unknown. Small molecules offer opportunities to both decode these cellular roles and develop therapeutics, however there are few examples of small molecules that target biologically relevant RNA tertiary structures. While RNA triple helices are a particularly attractive target, discovery of triple helix modulators has been hindered by the lack of correlation between small molecule affinity and effect on structural modulation, thereby limiting the utility of affinity-based screening as a primary filtering method. To address this challenge, we developed a high-throughput RT-qPCR screening platform that reports on the effect of mutations and additives, such as small molecules, on the stability of triple helices. Using the 3'-end of the oncogenic long non-coding RNA MALAT1 as a proof-of-concept, we demonstrated the applicability of both a two-step and a one-pot method to assess the impact of mutations and small molecules on the stability of the triple helix. We demonstrated the adaptability of the assay to diverse RNA tertiary structures by applying it to the SARS-CoV-2 pseudoknot, a key viral RNA structure recently identified as an attractive therapeutic target for the development of antivirals. Employment of a functional high-throughput assay as a primary screen will significantly expedite the discovery of probes that modulate the structural landscape of RNA structures and, consequently, help gain insight into the roles of these pervasive structures.
RNA三级结构在发现和表征方面呈指数级增长，这凸显了它们在多种人类疾病中的积极作用，然而其相互作用组和具体功能往往仍不为人所知。小分子为解码这些细胞作用和开发治疗方法提供了机会，然而，针对具有生物学相关性的RNA三级结构的小分子实例却很少。虽然RNA三链螺旋是一个特别有吸引力的靶点，但由于小分子亲和力与对结构调节的影响之间缺乏相关性，三链螺旋调节剂的发现受到了阻碍，从而限制了基于亲和力的筛选作为一种主要筛选方法的效用。为应对这一挑战，我们开发了一种高通量RT-qPCR筛选平台，该平台可报告突变和添加剂（如小分子）对三链螺旋稳定性的影响。以致癌长链非编码RNA MALAT1的3'端作为概念验证，我们展示了两步法和一锅法在评估突变和小分子对三链螺旋稳定性影响方面的适用性。通过将其应用于SARS-CoV-2假结（一种最近被确定为开发抗病毒药物有吸引力的治疗靶点的关键病毒RNA结构），我们证明了该检测方法对多种RNA三级结构的适应性。采用功能性高通量检测作为初步筛选将显著加快调节RNA结构构象的探针的发现，并因此有助于深入了解这些普遍存在的结构的作用。
