Small-Angle X‑ray\nScattering (SAXS) Combined\nwith SAXS-Driven Molecular Dynamics for Structural Analysis of Multistranded\nRNA Assemblies

Nucleic acids (RNA and DNA) play crucial roles in all\nliving organisms\nand find wide utility in clinical settings. The convergence of rationally\ndesigned nucleic acid multistranded assemblies with embedded therapeutic\nproperties has led to the development of a platform based on nucleic\nacid nanoparticles (NANPs). NANPs incorporate various functional moieties\nto deliver their combinations to diseased cells in a highly controlled\nmanner. Given that the structure and composition of NANPs can also\ninfluence their immunorecognition and biological activities, thorough\nverification of all designs is essential. We introduce an experimental\npipeline for small-angle X-ray scattering (SAXS) to gather structural\ndetails about the solution-state NANPs assembled from up to 12 RNA\nstrands. To the best of our knowledge, this study represents the largest\nmultistranded RNA nanoassemblies characterized in this manner to date.\nWe show that synchronized implementation of SAXS-driven molecular\ndynamics simulations reveals the diverse conformational landscape\ninhabited by these assemblies and provides insights into their immunorecognition.\nThe developed strategy expands the capabilities of therapeutic nucleic\nacids and emerging nucleic acid nanotechnologies.