Quantifying Structure‐Dependent Rigidity of 3D DNA Origami Boxes

DNA origami enables the programmable assembly of complex 3D nanostructures, yet their mechanical stability on solid surfaces remains insufficiently understood. Here, we present a comprehensive analysis of a hollow DNA origami box and its intermediate assembly states using atomic force microscopy (AFM) imaging in liquid, combined with finite element simulations. Beginning with the flat 6‐face sheet structure and progressing through partially folded intermediates to the fully enclosed box, we quantify how increasing structural completeness enhances nanoscale rigidity and resistance to deformation. AFM imaging shows that all nanostructures form as designed but are highly susceptible to tip‐induced collapse, with partially assembled intermediates undergoing complete flattening, while the fully closed box exhibiting only moderate deformation. Height and width measurements reveal reduced lateral spreading and increased vertical stability as additional faces are incorporated. Simulations reproduce the experimentally observed collapse behavior and demonstrate that the intrinsic hollowness of the DNA box renders it mechanically soft under load. Together, these results serve as guidelines linking structural enclosure and mechanical reinforcement to the stability of hollow DNA origami architectures, providing design guidelines for the development of more robust DNA origami‐based nanodevices as building blocks for intelligent nanoscale systems.