Group II introns are large self-splicing ribozymes. Since they exhibit considerable dynamic plasticity to attain catalytic activity, they serve as powerful model systems for studying RNA folding and splicing mechanisms. While being absent in the human genome, they occur in essential housekeeping genes of several human-pathogenic fungi and bacteria, making them promising antimicrobial targets with minimal host-cell toxicity.^[1] Intronistat B, a small-molecule inhibitor that binds the catalytic core, disrupts intron splicing.^[2] Although crystal structures provide static snapshots of inhibitor binding to the catalytic core, they offer valuable insights into the bound state. However, they do not fully capture how inhibition affects the structural rearrangements required for catalysis.^[3]

To address this limitation, we employ two-color single-molecule Förster Resonance Energy Transfer (smFRET) to follow structural rearrangement within the RNA. This approach allows us to follow conformational changes of individual RNA molecules during folding and catalysis, both in the presence and absence of Intronistat B. Our data reveal substantial alterations in domain-specific motions essential for catalytic activity, accompanied by pronounced changes in the dynamics of conformational rearrangements. These findings illuminate dynamic features of RNA when interacting with the small molecule that are otherwise inaccessible by static structural techniques, offering a foundation for the rational design of next-generation target-specific antimicrobials.

[1]        J. Nosek, M. Novotna, Z. Hlavatovicova, D. W. Ussery, J. Fajkus, L. Tomaska, Molecular Genetics and Genomics 2004, 272, 173

[2]        O. Fedorova, G. E. Jagdmann, R. L. Adams, L. Yuan, M. C. Van Zandt, A. M. Pyle, Nat. Chem. Biol. 2018, 14, 1073

[3]        I. Silvestri, J. Manigrasso, A. Andreani, N. Brindani, C. Mas, J.-B. Reiser, P. Vidossich, G. Martino, A. A. McCarthy, M. De Vivo, M. Marcia, Nat. Commun. 2024, 15, 4980