The large-scale analysis of small-molecule binding to diverse RNA structures is key to understanding the required interaction properties and selectivity for developing RNA-binding molecules toward RNA-targeted therapies. Here, we report a new system for performing the large-scale analysis of small molecule–RNA interactions using a multiplexed pull-down assay with RNA structure libraries. The system profiled the RNA-binding landscapes of G-clamp and thiazole orange derivatives, which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. We discuss the binding preferences of these molecules based on their large-scale affinity profiles. In addition, we selected combinations of fluorescent indicators and different ranks of RNA based on the information and screened for RNA-binding molecules using FID. RNAs with high- and intermediate-rank RNA provided reliable results. Our system provides fundamental information about small molecule–RNA interactions and facilitates the discovery of novel RNA-binding molecules.

对与不同 RNA 结构结合的小分子进行大规模分析是了解开发 RNA 结合分子以实现 RNA 靶向治疗所需的相互作用特性和选择性的关键。在这里，我们报告了一种新系统，用于使用 RNA 结构库进行多重下拉分析，对小分子与 RNA 相互作用进行大规模分析。该系统描绘了 G-clamp 和噻唑橙衍生物的 RNA 结合图谱，它们分别识别未配对的鸟嘌呤碱基，并且是荧光指示剂置换 (FID) 检测的良好探针。我们根据这些分子的大规模亲和力特征讨论了它们的结合偏好。此外，我们根据信息选择荧光指示剂和不同级别的RNA的组合，并使用FID筛选RNA结合分子。具有高等级和中等级 RNA 的 RNA 可提供可靠的结果。我们的系统提供有关小分子-RNA 相互作用的基本信息，并促进新型 RNA 结合分子的发现。