Bioproduction of ∼10 knt single-stranded DNA for constructing large DNA origami structures

folds have limitations in terms of functional integration and size expansion. Using longer scaffold strands can substantially increase the number of programmable functional sites and size of DNA origami. Therefore, preparing long single-stranded DNA (ssDNA) and stably synthesizing large DNA origami structures are key challenges. Here, we established a highly efficient biosynthesis platform for long ssDNA (∼10 knt) based on a phage-phagemid production system, and used this ssDNA to prepare large DNA origami structures with stable structure and high yield. The yield of the long ssDNA was increased by approximately an order of magnitude by optimizing the synthesis parameters in shake flasks and bioreactors. We successfully constructed large triangular (161 nm side length) and rectangular (93 × 115 nm) DNA origami structures, which have approximately 100 more potential functionalization sites compared to traditional structures based on M13mp18. This work contributes to improve the scalable production of long ssDNA scaffolds and provides a practical foundation for constructing large DNA origami nanostructures.