Uncovering Design and Assembly Rules for mRNA–DNA Origami

mRNA-DNA hybrid origami enables integration of the RNA functionality into programmable DNA nanostructures, yet robust design and assembly rules remain lacking. Here, we systematically define parameters governing the high-yield formation of compact mRNA-DNA hybrid origami. Using mature mRNAs encoding firefly luciferase, enhanced green fluorescent protein (EGFP), and mCherry as scaffolds, we designed five architectures spanning varied sizes, shapes, crossover geometries, and packing densities. We identify asymmetric A-form crossovers, monovalent-cation-rich buffers, and moderate-temperature annealing as critical for suppressing RNA degradation and kinetic trapping while accommodating RNA-DNA helical geometry. Atomic force microscopy confirms monodisperse, well-folded nanostructures with nanoscale precision comparable to that of DNA origami. These results establish generalizable design rules and a standardized synthesis protocol for mRNA-DNA hybrid origami.