Conformational dynamics at the pre-miR-377 Dicer site governs selective small-molecule recognition

Small molecules that target precursor microRNAs (pre-miRNAs) offer a strategy to modulate microRNA biogenesis, but rational design is limited by the intrinsic conformational dynamics of RNA. Here, we combine site-specific nuclear magnetic resonance (NMR) spectroscopy with enhanced-sampling molecular dynamics (MD) simulations to define the conformational ensemble of pre-miR-377 and its recognition by the small-molecule binder C1. Our data reveals that the Dicer cleavage site contains a dynamic adenosine bulge in which U25 shuffles between A3 and A4 with a ∼9:1 population ratio, generating two interconverting RNA triple structures. NMR-informed MD and binding simulations show that C1 engages this dynamic region through a pathway-dependent mechanism that favors minor-groove entry, stabilizes intermediate, partially reorganized Dicer-site conformations, and redistributes the conformational ensemble away from processing-competent states. A conformationally locked mutant abolishes binding, indicating that recognition depends on bulge-enabled RNA dynamics rather than a single static structure. These results define the structural and dynamic basis of small-molecule recognition at the pre-miR-377 Dicer site and highlight the importance of RNA conformational ensembles in RNA-targeted drug discovery.