A long-staple design approach towards the scalable production of scaffolded DNA origami

초록

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.

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