RNA structural plasticity underlies both biological regulation and opportunities for therapeutic targeting. We engineered a protein nanopore to function as a molecular ruler that reports RNA structural and kinetic responses at the single-molecule level. Using bulged RNA duplexes, we first established a direct correlation between helical bending and nanopore current amplitude, enabling quantitative readout of this three-dimensional RNA structural feature. Applying this principle to the U1 snRNA:5′ss duplex, a disease-relevant A-bulged splicing RNA implicated in spinal muscular atrophy, we found that the FDA-approved small molecule Risdiplam and its analog SMNC2 induce a novel blocking level that is consistent with a binding-driven conformational change. To understand the mechanism, we tested a U1 snRNA:5′ss mutant RNA that adopts an extended conformation. We discovered that ligand binding drives the structurally distinct mutant into a similar global conformation as that of the WT (wild-type). However, the duration of the bound states is significantly reduced compared to the WT. Our results suggest that local intermolecular interaction at the bulge determines the stability of the final bound conformation. By simultaneously resolving global structural features and binding kinetics, the nanopore uncovered how a small molecule can remodel the RNA conformational landscape, with application in structure-based RNA-targeting drug discovery.
Chen et al. (Sun,) studied this question.
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