Self-Assembly of Complex Nucleic Acid Nanostructures From Single-Stranded RNA Tiles

Programmed self-assembly using nucleic acids is a powerful method for producing nanostructures with precisely defined shapes and sizes. Recently, a new paradigm has emerged for constructing such structures using short DNA oligonucleotides or tiles. This single-stranded tile (SST) approach utilizes multiple nucleic acid strands each containing four binding sites that are complementary to the four nearest neighbors of the tile in the specified structure. This simple yet versatile construction motif has been successfully applied to fabricating complex one-, two-, and three-dimensional DNA architectures. SST nanostructures however have so far been limited to DNA, leaving RNA with its distinct structural, chemical, and biological properties completely unexplored as an SST building material. Here, we describe how the SST paradigm can be successfully applied to the assembly of complex RNA-based molecular architectures. These RNA SST structures are constructed from enzymatically synthesized RNA tiles that are subsequently pooled and assembled through thermal annealing in a one-pot process. We have used this approach to construct one-dimensional RNA nanoribbons and nanotubes with prescribed widths and diameters, respectively. Furthermore, we have assembled complex two-dimensional lattices with well-defined dimensions composed of up to 95 unique RNA tiles. These RNA nanostructures offer distinct biochemical properties compared to their DNA counterparts. Notably, we have found that RNA SST assemblies can be successfully processed by recombinant human dicer enzyme into siRNAs and offer the potential to encode dozens of siRNAs within a single nanostructured delivery vehicle.