Large-scale analysis of small molecule-RNA interactions using multiplexed RNA structure libraries

Abstract

Abstract 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 (TO and TO-3), which recognizes an unpaired guanine base and are good probes for fluorescent indicator displacement (FID) assays, respectively. Based on the information obtained from the bindings of TO and TO-3, we selected the combinations of fluorescent indicators and drug-targetable pre-miRNAs and screened for RNA-binding molecules using FID. Four hit compounds were identified, and three of them were validated. Our system provides fundamental information about small molecule–RNA interactions and facilitates the discovery of novel RNA-binding molecules.