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RNA is a natural multifunctional polymer, and is an essential component in both complex pathways and structures within the cellular environment. For this reason, artificial self-assembling RNA nanostructures are emerging as a powerful tool with broad applications in drug delivery and metabolic pathway regulation. To date, coordinated delivery of functional molecules via programmable RNA assemblies has been primarily done using nanosize RNA scaffolds. However, larger scaffolds could expand existing capabilities for spatial arrangement of ligands, and enable the controlled delivery of highly concentrated molecular loads. Here, we investigate whether micron-size RNA scaffolds can be assembled and further functionalized with different cargos (e.g. various siRNAs and fluorescent tags) for their synchronized delivery to diseased cells. Since known design approaches to build large RNA scaffolds are still underdeveloped, we apply a tiling method widely used in DNA nanotechnology. DNA tiles have been extensively used to build a variety of scalable and modular structures that are easily decorated with other ligands. Here, we adapt a double crossover (DX) DNA tile motif to design de novo DX RNA tiles that assemble and form lattices via programmed sticky end interactions. We optimize assembly protocols to guarantee high yield of RNA lattices. The resulting constructs are robust and modular with respect to the presence of distinct siRNAs and fluorophores. RNA tiles and lattices are successfully transfected in either human breast cancer or prostate cancer cells, where they efficiently knockdown the expression of target genes. Blood serum stability assays indicate that RNA lattices are more resilient to nuclease degradation when compared to individual tiles, thus making them better suited for therapeutic purposes. Overall, because of its design simplicity, we anticipate that this approach will be utilized for a wide range of applications in therapeutic RNA nanotechnology.

Programmable RNA microstructures for coordinated delivery of siRNAs