Nick-and-Digest Strategy for Programmable Circular ssDNA Production and Scalable DNA Origami Assembly

DNA origami enables the precise self-assembly of complex nanostructures with applications in drug delivery, biosensing, and nanoelectronics. However, the scalability of DNA origami is constrained by the limited length of the available single-stranded DNA (ssDNA) scaffolds. Here, we present a sequence-flexible "nick-and-digest" strategy to generate long circular ssDNA (cssDNA) scaffolds of customizable lengths directly from plasmid DNA. By combining Cas9 (D10A) nickase (Cas9n) with T7 exonuclease (T7 Exo), we generate high-purity cssDNA scaffolds of around 7,000 and 15,000 nucleotides (7k-nt and 15k-nt) with minimal sequence dependence. These extended scaffolds enable the one-pot folding of large-scale origami structures (147 × 107 nm) that double the surface area of conventional 7 kb designs. We optimize the denaturation temperature, annealing procedure, and staple-to-scaffold ratios to improve the folding efficiency while minimizing thermal damage. Compared to a two-step dimerization approach, the one-pot assembly achieves higher yield, fewer structural defects, and greater mechanical stability, as confirmed by atomic force microscopy (AFM) and coarse-grained molecular dynamics (one-pot: -15.19 ± 0.014<i>k</i> _B <i>T</i>; two-step: -13.46 ± 0.034<i>k</i> _B <i>T</i>). Our work provides a robust method for generating long cssDNA and optimizing large-scale DNA origami assembly, overcoming key barriers in scalability and programmability. This approach opens new avenues for applications in large-scale nanoelectronics, high-density data storage, and advanced therapeutic platforms.