Hapten-Decorated DNA Nanostructures Decipher the Antigen-Mediated Spatial Organization of Antibodies Involved in Mast Cell Activation

[Image: see text] The immunological response of mast cells is controlled by the multivalent binding of antigens to immunoglobulin E (IgE) antibodies bound to the high-affinity receptor FcεRI on the cell membrane surface. However, the spatial organization of antigen–antibody–receptor complexes at the nanometer scale and the structural constraints involved in the initial events at the cell surface are not yet fully understood. For example, it is unclear what influence the affinity and nanoscale distance between the binding partners involved have on the activation of mast cells to degranulate inflammatory mediators from storage granules. We report the use of DNA origami nanostructures (DON) functionalized with different arrangements of the haptenic 2,4-dinitrophenyl (DNP) ligand to generate multivalent artificial antigens with full control over valency and nanoscale ligand architecture. To investigate the spatial requirements for mast cell activation, the DNP–DON complexes were initially used in surface plasmon resonance (SPR) analysis to study the binding kinetics of isolated IgE under physiological conditions. The most stable binding was observed in a narrow window of approximately 16 nm spacing between haptens. In contrast, affinity studies with FcεRI-linked IgE antibodies on the surface of rat basophilic leukemia cells (RBL-2H3) indicated virtually no distance-dependent variations in the binding of the differently structured DNP–DON complexes but suggested a supramolecular oligovalent nature of the interaction. Finally, the use of DNP–DON complexes for mast cell activation revealed that antigen-directed tight assembly of antibody-receptor complexes is the critical factor for triggering degranulation, even more critical than ligand valence. Our study emphasizes the significance of DNA nanostructures for the study of fundamental biological processes.