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.