Nanopore Discrimination of Protein–Small-Molecule Drug Complexes at Near-Atomic Resolution

Nanopore is a single-molecule technology for sensing biomolecules. Biomolecular interactions are essential biological processes that govern biological functions and therapeutic responses. However, high-resolution nanopore sensing of biomolecular interactions, such as protein-ligand interactions, remains challenging. In this study, we demonstrate that a YaxAB nanopore with LiCl-modulated electrostatic potential enables detection of molecular interactions of the BRD4 protein with histone peptides, as well as diverse small-molecule drugs, at the single-molecule level. Our electrical recordings and molecular dynamics simulations confirm that the oscillating dynamics of BRD4 within the funneled YaxAB nanopore generate two-level current transitions between narrow- and wide-pore regions. Using the parameters derived from dual-level dynamics and their signal decomposition, a YaxAB nanopore sensing approach enables the sensitive discrimination of BRD4-small-molecule drug complexes with a subtle mass difference as small as 2.5 Da. This near-atomic, high-resolution sensing capability of YaxAB nanopores may enable applications in single-molecule-based drug discovery, proteomics, and diagnostics.