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

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.