Keynote Abstract

Designing Translation-competent mRNA Origami Nanoparticles within the OxRNA Ecosystem

OxDNA and oxRNA are coarse-grained models specifically designed for DNA and RNA nanotechnology. The models capture basic thermodynamic, mechanical and structural properties of single-stranded and double-stranded DNA / RNA. In addition to the models, we develop an associated ecosystem of design and analysis tools, including the interactive design tool oxView. We give an overview of the latest additions to the oxDNA ecosystem and present our application of model-driven design of a single-stranded RNA origami nanostructure. mRNAs with highly structured ORFs have superior cold-chain stability and in vivo translation, but the boundary between compact folding and efficient ribosome access remains unclear. Single-stranded mRNA origami (mRNA-OG) is an intramolecular folding approach producing highly double-stranded nanoparticles and offers a route to extreme transcript compaction, but its feasibility hinges on whether cellular translation machinery can effectively access and unwind the transcript without activating cellular stress pathways. We show that our designed mRNA-OG nanoparticles adopt compact, planar architectures with exceptional robustness to variation in ionic strength and temperature. Despite their dense folding, these constructs remain translation-competent in both immortalized cell lines and primary cells. Compared with unstructured transcripts, mRNA-OGs display markedly improved stability during 4 C storage and show no activation of integrated dsRNA-sensing pathways, as assessed by polysome profiling and EIF2A phosphorylation assays. Our results demonstrate that model-driven rational design of mRNA secondary structure provides a stepping stone toward stable, cell type-addressable, and potentially self-adjuvanting mRNA therapeutics.