Dynamic Binder Exchange Improves Protein Labeling Efficiency in DNA‐PAINT up to 15‐Fold

Abstract

Abstract Dynamic Binder Exchange (DyBE) enhances DNA‐PAINT (Point Accumulation for Imaging in Nanoscale Topography) super‐resolution microscopy by exploiting transient, reversible binder‐target interactions. DyBE uses DNA‐conjugated binders such as nanobodies as both targeting and docking moieties, integrating their characteristic higher off‐rates with DNA‐PAINT blinking to efficiently sample target sites. This dual‐kinetic scheme increases labeling efficiency up to 15‐fold, enabling sensitive detection of targets previously inaccessible due to limitations of high‐off‐rate binders. Using DyBE, the study reveals pre‐existing HER2 homodimers and ligand‐induced EGFR‐HER2 heterodimers at single‐protein resolution with high fidelity. DyBE expands the usable binder repertoire, advancing spatial proteomics and enabling mechanistic drug studies of receptor organization and signaling.