Programmable multispecific DNA-origami-based T-cell engagers.

Multispecific antibodies have emerged as versatile therapeutic agents, and therefore, approaches to optimize and streamline their design and assembly are needed. Here we report on the modular and programmable assembly of IgG antibodies, F(ab) and scFv fragments on DNA origami nanocarriers. We screened 105 distinct quadruplet antibody variants in vitro for the ability to activate T cells in the presence of target cells. T-cell engagers were identified, which in vitro showed the specific and efficient T-cell-mediated lysis of five distinct target cell lines. We used these T-cell engagers to target and lyse tumour cells in vivo in a xenograft mouse tumour model. Our approach enables the rapid generation, screening and testing of bi- and multispecific antibodies to facilitate preclinical pharmaceutical development from in vitro discovery to in vivo proof of concept.

Revealing the structures of megadalton-scale DNA complexes with nucleotide resolution.

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.

Exploring Nucleosome Unwrapping Using DNA Origami.

We establish a DNA origami based tool for quantifying conformational equilibria of biomolecular assemblies as a function of environmental conditions. As first application, we employed the tool to study the salt-induced disassembly of nucleosome core particles. To extract binding constants and energetic penalties, we integrated nucleosomes in the spectrometer such that unwrapping of the nucleosomal template DNA, leading from bent to more extended states was directly coupled to the conformation of the spectrometer. Nucleosome unwrapping was induced by increasing the ionic strength. The corresponding shifts in conformation equilibrium of the spectrometer were followed by direct conformation imaging using negative staining TEM and by FRET read out after gel electrophoretic separation of conformations. We find nucleosome dissociation constants in the picomolar range at low ionic strength (11 mM MgCl2), in the nanomolar range at intermediate ionic strength (11 mM MgCl2 with 0.5-1 M NaCl) and in the micromolar range at larger ionic strength (11 mM MgCl2 with ≥1.5 M NaCl). Integration of up to four nucleosomes stacked side-by-side, as it might occur within chromatin fibers, did not appear to affect the salt-induced unwrapping of nucleosomes. Presumably, such stacking interactions are already effectively screened at the nucleosome unwrapping conditions. Our spectrometer provides a modular platform with a direct read out to study conformational equilibria for targets from small biomolecules up to large macromolecular assemblies.

Uncovering the forces between nucleosomes using DNA origami.

Revealing the energy landscape for nucleosome association may contribute to the understanding of higher-order chromatin structures and their impact on genome regulation. We accomplish this in a direct measurement by integrating two nucleosomes into a DNA origami-based force spectrometer, which enabled subnanometer-resolution measurements of nucleosome-nucleosome distance frequencies via single-particle electron microscopy imaging. From the data, we derived the Boltzmann-weighted distance-dependent energy landscape for nucleosome pair interactions. We find a shallow but long-range (~6 nm) attractive nucleosome pair potential with a minimum of -1.6 kcal/mol close to direct contact distances. The relative nucleosome orientation had little influence, but histone H4 acetylation or removal of histone tails drastically decreased the interaction strength. Because of the weak and shallow pair potential, higher-order nucleosome assemblies will be compliant and experience dynamic shape fluctuations in the absence of additional cofactors. Our results contribute to a more accurate description of chromatin and our force spectrometer provides a powerful tool for the direct and high-resolution study of molecular interactions using imaging techniques.

Placing molecules with Bohr radius resolution using DNA origami.

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.