Folding Competition and Dynamic Transformation in DNA Origami: Parallel Versus Antiparallel Crossovers

Abstract DNA is a versatile abiomaterial for constructing nanostructures with biomedical and biotechnological applications. Among the methods available, DNA origami is a robust and widely recognized technique. Traditionally, most origami designs adopt antiparallel crossovers in both scaffold and staple strands, with less emphasis on parallel crossovers, which offer advantages like enhanced nuclease resistance and single‐strand routing potential. Here, a DNA origami nanostructure is designed, featuring two rotational panels that can be locked into configurations based on either antiparallel or parallel crossovers. By systematically varying the length and arrangement of these key staples, 36 pairs of antiparallel and parallel designs are studied in competitive folding tests, providing insights into the relative preference for each design. The 12 antiparallel and parallel designs are ranked, their folding pathways are examined, and nuclease resistance is assessed. The results reveal that the arrangement of staples near the central scaffold crossover is crucial for shifting between parallel and antiparallel conformations. Additionally, a two‐way isothermal transformation between antiparallel and parallel origami driven by toehold‐mediated displacement reactions is demonstrated, highlighting the potential of parallel designs as dynamic nanodevices for temperature‐sensitive environments. This study offers valuable insights into ‐ dynamics in antiparallel and parallel DNA origami, opening opportunities for designing nanodevices based on parallel crossovers.