Single-Molecule Mapping Landscape of Multivalent Antibody-DNA Framework Conjugates

Patterned assembly of multivalent antibody complexes using DNA nanostructure templates holds the potential for advancing studies of cellular signaling and smart theranostic applications. However, evaluating the heterogeneity in protein conjugation efficiency at distinct sites on DNA templates remains challenging. Here, we utilize atomic force microscopy to measure the coupling of antibodies at various positions on two-dimensional rectangular DNA origami frameworks at the single-molecule level, generating spatial maps of antibody binding efficiencies across the structures. We observe that a discrete distribution of docking sites (spacing of at least 18 nm) on the framework leads to a progressive decrease in the antibody coupling efficiency from the periphery toward the center. In contrast, a continuous distribution of docking sites (spacing of ∼10 nm) results in a higher efficiency at the center relative to the periphery. We reason that the two opposing trends result from trade-offs among Coulombic repulsion, steric hindrance, and multivalent cooperative effects. This study presents a quantitative evaluation tool for protein-DNA framework conjugates, providing insights into optimizing DNA framework-based systems for improved precision in diagnostics and therapeutic applications.