BPS2026 – Toward a design framework for chemically modified RNA: Integrating cryo-EM, functional assays, and molecular dynamics

In vitro transcription with chemically modified nucleotides enables the production of large amounts of non-natural RNA with enhanced biological properties, yet how such modifications influence the folding and dynamics of complex RNA nanostructures remains poorly understood. As this field advances, RNA-focused molecular dynamics (MD) simulations are emerging as a valuable complement to experimental methods, offering atomistic insight into structural dynamics often inaccessible to cryo-electron microscopy (cryo-EM) alone. We used MD simulations to examine the effects of incorporating 2′-fluoro-modified pyrimidines (2′-FY) in RNA origami scaffolds from individual RNA motifs to full RNA-protein assemblies. At the motif level, simulations showed that 2′-FY disrupted stabilizing interactions in tetraloops, weakened helix-helix contacts in crossovers, and increased flexibility in kissing loops. Extending this analysis to several full origami scaffolds revealed that these local perturbations can propagate to affect global structure and dynamics, with some modified structures adopting distinct conformational landscapes compared to their unmodified counterparts. Simulations of an RNA origami “pointer” bearing a 2′-FY aptamer bound to the SARS-CoV-2 spike protein indicated that 2′-FY stabilizes the aptamer-receptor binding domain (RBD) interface by reducing loop flexibility and enhancing local contacts, compared to the unmodified RNA version. These simulation-based insights aligned with experimental observations. Cryo-EM showed that the overall architectures of several 2′-FY RNA origami scaffolds were largely retained. In addition, while the unmodified RNA aptamer showed no detectable binding to the SARS-CoV-2 spike protein, cryo-EM resolved the 2′-FY aptamer-RBD interface at 3.4 Å, supporting the stabilizing effect seen in simulations. Together, this integrative approach shows how combining cryo-EM, functional assays, and MD simulations can reveal how chemical modifications reshape the structural landscape of both RNA motifs and large origami scaffolds, providing a framework for the rational design of modified RNA nanostructures.