Label-Free Mapping of Multivalent Binding Pathways with Ligand–Receptor-Anchored Nanopores

Understanding single-molecule multivalent ligand–receptor interactions is crucial for comprehending molecular recognition at biological interfaces. However, label-free identifications of these transient interactions during multistep binding processes remains challenging. Herein, we introduce a ligand–receptor-anchored nanopore that allows the protein to maintain structural flexibility and favorable orientations in native states, mapping dynamic multivalent interactions. Using a four-state Markov chain model, we clarify two concentration-dependent binding pathways for the Omicron spike protein (Omicron S) and soluble angiotensin-converting enzyme 2 (sACE2): sequential and concurrent. Real-time kinetic analysis at the single–monomeric subunit level reveals that three S1 monomers of Omicron S exhibit a consistent and robust binding affinity toward sACE2 (−13.1 ± 0.2 kcal/mol). These results highlight the enhanced infectivity of Omicron S compared to other homologous spike proteins (WT S and Delta S). Notably, the preceding binding of sACE2 to Omicron S facilitates the subsequent binding steps, which was previously obscured in bulk measurements. Our single-molecule studies resolve the controversy over the disparity between the measured spike protein binding affinity with sACE2 and the viral infectivity, offering valuable insights for drug design and therapies.