Abstract
Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg2+ concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.