Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings

The internal design of DNA nanostructures defines how they behave in different environmental conditions, such as endonuclease-rich or low-Mg²⁺ solutions. Notably, the inter-helical crossovers that form the core of such DNA objects have a major impact on their mechanical properties and stability. Importantly, crossover design can be used to optimize DNA nanostructures for target applications, especially when developing them for biomedical environments. To elucidate this, two otherwise identical DNA origami designs are presented that have a different number of staple crossovers between neighboring helices, spaced at 42- and 21- basepair (bp) intervals, respectively. The behavior of these structures is then compared in various buffer conditions, as well as when they are exposed to enzymatic digestion by DNase I. The results show that an increased number of crossovers significantly improves the nuclease resistance of the DNA origami by making it less accessible to digestion enzymes but simultaneously lowers its stability under Mg²⁺-free conditions by reducing the malleability of the structures. Therefore, these results represent an important step toward rational, application-specific DNA nanostructure design.