Adam Wier (University of Notre Dame)

Abstract:   Electrophilic small-molecules have enabled ligand discovery for difficult-to-drug proteins; however, there have been few uses of reactive handles in RNA-targeted ligand discovery. The authors of this paper establish one of the first examples of an electrophilic small-molecule ligand for an RNA target, the PreQ1 riboswitch in Thermoanaerobacter tengcongensis (Tt). By introducing a propylbromide group onto a previously known non-covalent ligand, the authors demonstrate a strategy for electrophilic derivation of existing scaffolds, known as “tethering”. Monitoring their ligands’ reactivities with an in vitro construct by anion-exchange high performance liquid chromatography (AE-HPLC), the authors test different leaving groups, optimize their linker lengths, and parse the selectivity of their Brc₃DPQ₁ ligand among similar bacterial PreQ₁ riboswitches. Using atomic mutagenesis, the authors validate a guanine-N7 as the reacting nucleophile in the RNA and elucidate its necessity over other nucleophilic bases. The authors then demonstrate the ability of Brc₃DPQ₁ to target the Tt PreQ1 riboswitch in vivo using a GFP construct. To expand in vivo utility, they demonstrate the utility of tetherable fluorophores to enable higher-resolution covalent fluorescent light-up aptamers (coFLAPs), identifying MsOc₃HBC as a ligand for the Pepper aptamer. This coFLAP system is shown to be valuable for cellular localization studies of transcripts, including in fluorescence recovery after photobleaching (FRAP). Finally, the authors demonstrate the potential for their tethering method to identify new covalent drugs for RNA, optimizing a non-native dibenzofuran ligand for higher-potency binding to the Tt preQ₁ riboswitch. This paper lays the foundation for covalent ligand discovery in RNA chemical biology.

Authors: Raphael Bereiter¹, Laurin Flemmich¹, Kamila Nykiel², Sarah Heel¹, Stephan Geley³, Malou Hanisch², Clemens Eichler¹, Kathrin Breuker¹, Alexandra Lusser², & Ronald Micura¹

¹Institute of Organic Chemistry and Center for Molecular Biosciences (CMBI), University of Innsbruck, Innsbruck, Austria

²Institute of Molecular Biology and ³Institute of Pathophysiology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

Nature Chemical Biology. 2025. DOI: https://doi.org/10.1038/s41589-024-01801-3