Electron microscopy with a DNA origami force spectrometer reveals the energy landscapes for nucleosome-nucleosome interactions.

Molecular self-assembly with nucleic acids can be used to fabricate discrete objects with defined sizes and arbitrary shapes. It relies on building blocks that are commensurate to those of biological macromolecular machines and should therefore be capable of delivering the atomic-scale placement accuracy known today only from natural and designed proteins. However, research in the field has predominantly focused on producing increasingly large and complex, but more coarsely defined, objects and placing them in an orderly manner on solid substrates. So far, few objects afford a design accuracy better than 5 nm, and the subnanometre scale has been reached only within the unit cells of designed DNA crystals. Here, we report a molecular positioning device made from a hinged DNA origami object in which the angle between the two structural units can be controlled with adjuster helices. To test the positioning capabilities of the device, we used photophysical and crosslinking assays that report the coordinate of interest directly with atomic resolution. Using this combination of placement and analysis, we rationally adjusted the average distance between fluorescent molecules and reactive groups from 1.5 to 9 nm in 123 discrete displacement steps. The smallest displacement step possible was 0.04 nm, which is slightly less than the Bohr radius. The fluctuation amplitudes in the distance coordinate were also small (±0.5 nm), and within a factor of two to three of the amplitudes found in protein structures.

The methods of DNA nanotechnology enable the rational design of custom shapes that self-assemble in solution from sets of DNA molecules. DNA origami, in which a long template DNA single strand is folded by many short DNA oligonucleotides, can be employed to make objects comprising hundreds of unique DNA strands and thousands of base pairs, thus in principle providing many degrees of freedom for modelling complex objects of defined 3D shapes and sizes. Here, we address the problem of accurate structural validation of DNA objects in solution with cryo-EM based methodologies. By taking into account structural fluctuations, we can determine structures with improved detail compared to previous work. To interpret the experimental cryo-EM maps, we present molecular-dynamics-based methods for building pseudo-atomic models in a semi-automated fashion. Among other features, our data allows discerning details such as helical grooves, single-strand versus double-strand crossovers, backbone phosphate positions, and single-strand breaks. Obtaining this higher level of detail is a step forward that now allows designers to inspect and refine their designs with base-pair level interventions.