Super-selective multivalent ligand–receptor interactions display a signature step-like onset in binding when meeting a characteristic density of target receptors. Materials engineered for super-selective binding generally display a high number of flexible ligands to enhance the systems’ avidity. In many biological processes, however, ligands are present in moderate copy numbers and arranged in spatio-temporal patterns. In this low-valency regime, the rigidity of the ligand-presenting architecture plays a critical role in the selectivity of the multivalent complex through decrease of the entropic penalty of binding. Exploiting the precision in spatial design inherent to the DNA nanotechnology, we engineered a library of rigid architectures to explore how valency, affinity, and nano-spacing control the presence of super-selectivity in multivalent binding. A micromolar monovalent affinity was required for super-selective binding to be observed within low-valency systems, and the transition point for stable interactions was measured at hexavalent ligand presentation, setting the limits of the low-valency regime. Super-selective binding was observed for all hexavalent architectures, and, more strikingly, the ligand pattern determined the selectivity onset. Hereby, we demonstrate for the first time that nano-control of geometric patterns can be used to discriminate between receptor densities in a super-selective manner. Materials that were indistinguishable in their molecular composition and ligand valency bound with various efficacies on surfaces with constant receptor densities. We define this new phenomenon in super-selective binding as multivalent pattern recognition.

Nucleic acids and lipids function in close proximity in biological processes, as well as in nanoengineered constructs for therapeutic applications. As both molecules carry a rich charge profile, and frequently...

Targeting cells specifically based on receptor expression levels remains an area of active research to date. Selective binding of receptors cannot be achieved by increasing the individual binding strength, as this does not account for differing distributions of receptor density across healthy and diseased cells. Engaging receptors above a threshold concentration would be desirable in devising selective diagnostics. Integrins are prime target candidates as they are readily available on the cell surface and have been reported to be overexpressed in diseases. Insights into their spatial organization would therefore be advantageous to design selective targeting agents. Here, we investigated the effect of activation method on integrin α5β1 clustering by immunofluorescence and modeled the global neighbor distances with input from an immuno-staining assay and image processing of microscopy images. This data was used to engineer spatially-controlled DNA-scaffolded bivalent ligands, which we used to compare trends in spatial-selective binding observed across HUVEC, CHO and HeLa in resting versus activated conditions in confocal microscopy images. For HUVEC and CHO, the data demonstrated an improved selectivity and localisation of binding for smaller spacings ~7 nm and ~24 nm, in good agreement with the model. A deviation from the mode predictions for HeLa was observed, indicative of a clustered, instead of homogeneous, integrin organization. Our findings demonstrate how low-technology imaging methods can guide the design of spatially controlled ligands to selectively differentiate between cell type and integrin activation state.

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