Efficient Self-Assembly and Monodispersity Control of 24-Helix DNA Origami Structures

DNA origami technology enables the precise assembly of two-and three-dimensional nanostructures with atomic-level accuracy through the programmed annealing of a single-stranded DNA scaffold and staple strands, demonstrating significant potential in biosensing, nanophotonics, and targeted drug delivery. However, nonspecific aggregation among origami structures severely limits their functionality. In this study, we employed the 24-helix bundle (24HB) DNA origami as a model system and successfully achieved a structurally intact product yield of 96% by optimizing the synthesis conditions, including Mg²⁺ concentration and annealing gradient. Furthermore, we investigated the effects of two strategies—terminal single-strand brush modification and scaffold loop design—on the monodispersity of the 24HB DNA origami. The results demonstrated that modifying the 24HB DNA origami with PolyT (4 nt or 7 nt) single-strand brushes at one terminus significantly improved monodispersity, with the 7 nt brush exhibiting superior dispersion efficiency compared with the 4 nt variant. Additionally, a double-end scaffold loop design (72% monodispersity) outperformed the single-end design (42%) and was comparable to the 7 nt single-end PolyT brush modification (78%). Finally, by integrating a double-end scaffold loop with terminal single-strand brushes, we developed a composite strategy that achieved 87% monodispersity. This study establishes a standardized protocol for high-purity DNA origami fabrication and provides universal design principles for the precise assembly of functional DNA nanodevices.