Scaffolded DNA origami enables the programmable construction of nanoscale structures through the hybridization of a long single-stranded scaffold with hundreds of short staple strands. However, the reliance on numerous synthetic oligonucleotides remains a key barrier to scalable and cost-effective production of DNA nanostructures. In this study, we introduce a long-staple design strategy that extends the length of individual staple strands to 100–200 nucleotides (nt), thereby reducing the total number of strands required while maintaining assembly efficiency and structural fidelity. We demonstrate that this approach is broadly compatible with a variety of origami architectures, including both manually designed lattice-based structures and algorithmically generated wireframe geometries, without requiring changes to well-established design workflows. Using representative 2D and 3D structures, we show that long staples can assemble efficiently under the same thermal annealing conditions and Mg2+ concentrations as short staples, yielding final structures with comparable morphology. To further support biological production of staple strands, we generated long staples via rolling circle amplification (RCA) using custom-designed circular templates, each encoding a specific long staple sequence. This modular design allows for flexible and selective synthesis of desired staples, either individually or in pooled formats. These RCA-derived staples were successfully used in structure assembly, confirming the feasibility of enzyme-based synthesis for long-staple designs. This modular and adaptable strategy offers a practical route toward scalable fabrication of functional DNA nanostructures across diverse design frameworks.
