Toward high-density streptavidin arrays on DNA origami nanostructures

The binding of the protein streptavidin (SAv) to biotin-modified DNA origami nanostructures (DONs) is widely employed in the single-molecule study of chemical reactions, the arrangement of functional proteins and nanomaterials with molecular precision, and numerous instances of DON-based cryptography, steganography, and computing. The latter application areas in particular would benefit from high-density SAv arrays to achieve higher information densities. The finite size and tetrameric nature of SAv, however, pose certain limits to the SAv density that can be achieved in such arrays. In this work, we explore these limits by investigating the impact of selected design factors and environmental conditions on SAv binding to DON-supported biotin arrays. We identify the optimal distance between neighboring binding sites and the optimal length of the single-stranded spacer between DON surface and biotin modification. This allows us to assemble a 2D SAv array composed of 20 biotin modifications arranged at a density of about 0.008 nm-2 with an average SAv-Bt binding yield of about 70 %. Since higher binding yields are achieved for equivalent 1D arrays, our results suggest that molecular crowding is a major factor that limits the maximum binding yield achievable in such 2D arrays.