Design And Modeling Of A Tetrahedron Nanostructure For Enhanced Delivery Of Rnai Substrates

RNA has been established as a flexible material for constructing nanoparticles, allowing for programmable, precise control of their shape and size. In addition, it allows for functional extensions of the structural scaffolds with multiple RNA interference (RNAi) substrates or other RNA or RNA/DNA hybrid units. We present a new nanoscaffold that brings together building blocks of previously tested nanorings with additional motifs that facilitate linking hexameric faces together into a truncated tetrahedron. This presentation focuses on the modular nature of the tetrahedron design, its modeling and computational characterization, which prompted further development of RNA nanostructure modeling software. Molecular dynamics (MD) characterization of the tetrahedron model showed a stable scaffold with limited flexing. Modeling is in excellent agreement with the experimentally measured particle diameter based on dynamic light scattering (DLS) and atomic force microscopy (AFM) sizing. The original energy-minimized model fits the cryoEM density envelope very well. MD-predicted low levels of the full structure distortions and potentially most flexible areas are also consistent with the cryoEM results. Taken together, computational and experimental characterizations agree with each other in supporting the physical stability and shape of the structure. These characteristics appear to be important to the viability of the design as a delivery vehicle. The tetrahedral scaffold exhibits improved RNAi efficacy of its Dicer substrate siRNAs compared to similar RNA-based scaffolds. The current design can deliver up to 12 functionalities at once. Enhanced cellular uptake and processivity appear to be related to the shape of this nanostructure. Experimental characterization results strongly suggest that considering the physical characteristics of RNA nanoscaffolds should be an integral part of the design process of functional RNA platforms. Funded in part by HHSN261200800001E