In a model system of triangular DNA origami subunits that assemble into tubules, the authors use cryo-EM to image the various conformations of two subunits connected by six single-stranded DNA sticky ends. The more flexible interparticle interactions dominate the mechanical response over the relatively rigid triangular subunits, which would not be captured in the characterization of single subunits. Flash freezing the dimeric assemblies traps them in their equilibrated states, and the microscopy images are used to reconstruct their average structure. REgularized LIkelihood OptimizatioN (RELION) multi-body refinement analysis calculates the principal components of their motion, which are used to generate individual fluctuations. This analysis results in a dynamical matrix that contains mechanical information about the dimer such as translation, roll, twist and bends. The range of these angular deformations can be visualized (pictured).
The information from the dimer-level characterization can be used to inform the assembly at a larger scale. These DNA origami subunits are designed to assemble into achiral tubules that are ten monomers in circumference. However, fluctuations in the binding angles can cause variation during assembly leading to a distribution of chiral and achiral tubules with varied diameters, which is not accurately captured by simple elastic models. Using their cryo-EM multi-body refinement method, Rogers and colleagues extract bending angles and preferred average displacements for each binding side of the triangle. This information is used in additional simulations to find the minimum energy configuration for individual subunits. The authors develop a collective elastic energy model that more accurately reflects the experimentally observed tubule states. Differences between the binding angle in the assembled tubules compared with the dimers imply that multiple subunits must cooperatively interact and reorient when forming the superstructure assemblies. With these insights in hand, the authors redesign the initial subunit to correct for twist. These twist corrections allow for the self-assembly of the desired tubules with high fidelity.
This is a preview of subscription content, access via your institution
