Uncovering Design and Assembly Rules for mRNA–DNA Origami

mRNA–DNA hybrid origami offers a powerful route to combine the structural programmability of DNA origami with the biological functionality of messenger RNA, but generalizable design and assembly rules for these hybrids remain poorly defined. Here we systematically investigate the design principles and synthesis conditions that govern high-yield formation of mRNA–DNA hybrid nanostructures. Using mature mRNAs encoding firefly luciferase, EGFP, and mCherry as scaffolds, we construct a series of five hybrid compact origamis with diverse sizes, shapes, crossover strategies, and packing densities. We identify key parameters that control folding fidelity, including asymmetric A-form crossovers, monovalent-cation concentrations, and moderate-temperature annealing protocols, which together mitigate RNA instability, reduce kinetic traps, and accommodate RNA–DNA helical geometry. Atomic force microscopy reveals monodisperse, well-folded structures consistent with design expectations across most architectures and confirms that optimized conditions produce nanoscale precision comparable to DNA origami. Our findings establish generalizable design rules and a standard synthesis protocol for mRNA–DNA hybrid origami, providing a framework for their use in gene delivery and other RNA-based nanotechnologies.