Barcoded DNA origami structures for multiplexed optimization and enrichment of DNA-based protein-binding cavities.

Simultaneous binding of molecules by multiple binding partners is known to strongly reduce the apparent dissociation constant of the corresponding molecular complexes, and can be used to achieve strong, non-covalent molecular interactions. Based on this principle, efficient binding of proteins to DNA nanostructures has been achieved previously by placing several aptamers in close proximity to each other onto DNA scaffolds. Here, we develop an approach for exploring design parameters, such as the geometric arrangement or the nanomechanical properties of the binding sites, that use two-dimensional DNA origami-based nanocavities that bear aptamers with known mechanical properties at defined distances and orientations. The origami structures are labelled with barcodes, which enables large numbers of binding cavities to be investigated in parallel and under identical conditions, and facilitates a direct and reliable quantitative comparison of their binding yields. We demonstrate that binding geometry and mechanical properties have a dramatic effect on origami-based multivalent binding sites, and that optimization of linker spacings and flexibilities can improve the effective binding strength of the sites substantially.

Orthogonal Protein Assembly on DNA Nanostructures Using Relaxases.

DNA-binding proteins are promising reagents for the sequence-specific modification of DNA-based nanostructures. Here, we investigate the utility of a series of relaxase proteins-TrwC, TraI, and MobA-for nanofunctionalization. Relaxases are involved in the conjugative transfer of plasmids between bacteria, and bind to their DNA target sites via a covalent phosphotyrosine linkage. We study the binding of the relaxases to two standard DNA origami structures-rodlike six-helix bundles and flat rectangular origami sheets. We find highly orthogonal binding of the proteins with binding yields of 40-50 % per binding site, which is comparable to other functionalization methods. The yields differ for the two origami structures and also depend on the position of the binding sites. Due to their specificity for a single-stranded DNA target, their orthogonality, and their binding properties, relaxases are a uniquely useful addition to the toolbox available for the modification of DNA nanostructures with proteins.

Surface-assisted large-scale ordering of DNA origami tiles.

The arrangement of DNA-based nanostructures into extended higher order assemblies is an important step towards their utilization as functional molecular materials. We herein demonstrate that by electrostatically controlling the adhesion and mobility of DNA origami structures on mica surfaces by the simple addition of monovalent cations, large ordered 2D arrays of origami tiles can be generated. The lattices can be formed either by close-packing of symmetric, non-interacting DNA origami structures, or by utilizing blunt-end stacking interactions between the origami units. The resulting crystalline lattices can be readily utilized as templates for the ordered arrangement of proteins.