Robust Accessibility of Addressable Sites in Defective DNA Origami

Owing to its unique programmability and addressability, DNA origami-based nanofabrication has been widely utilized in fields such as nanomedicine and nanophotonics. The accessibility of addressable sites on DNA origami structures is crucial for their use as nanofabrication platforms. In this study, we systematically investigated the impact of structural defects on accessibility using a classic six-helix bundle (6HB) DNA origami and two variants with subsets of DNA staple strands deleted, introducing programmable defects in 6HB nanostructures. DNA point accumulation for imaging in nanoscale topography super-resolution microscopy was employed to monitor hybridization and dehybridization at each addressable site and analyze corresponding localizations and kinetics. Statistical analysis revealed that addressable sites on 6HB nanostructures retained significant accessibility robustness despite structural defects, which was further supported by molecular dynamics simulations. These results provide valuable insights into the design principles and applications of DNA origami.