Ligand specificity and adaptability revealed by the first Guanine-II riboswitch tertiary structure

A comprehensive understanding of the fundamental principles governing RNA–small molecule interactions is crucial for advancing RNA-targeting therapeutics with small molecules. Riboswitches, a class of noncoding RNAs, regulate gene expression by direct interaction with small-molecule metabolites. In this work, we report an in-depth structure-based investigation of a newly identified riboswitch, Guanine-II, which, despite sharing a conserved scaffold with the Guanine-I riboswitch, exhibits strikingly distinct small molecule ligand-binding characteristics. Through a comprehensive structural analysis of the Guanine-II riboswitch bound to various guanine analogs, combined with comparative studies of other guanine riboswitch variants, including Guanine-I and Xanthine-II riboswitches, as well as isothermal titration calorimetry, we reveal local structural rearrangements that precisely modulate small-molecule ligand adaptability. We further demonstrate that subtle differences in the composition and peripheral architecture of the binding pocket are key determinants of ligand-binding specificity. Additionally, based on the similarity in ligand recognition patterns with the tetrahydrofolate-II riboswitch, we identified additional compounds that bind to the Guanine-II riboswitch through a structure-guided rational search, providing valuable structural insights for the discovery of small molecules targeting RNA.