Stoichiometrically Defined Antibody–DNA Conjugates for Quantitative Super-Resolution Imaging

DNA-PAINT enables super-resolution imaging with molecular-scale precision and, through qPAINT analysis, quantitative determination of protein copy numbers. However, qPAINT accuracy critically depends on the stoichiometric definition of the DNA-antibody conjugate. Conventional amine-reactive labeling generates heterogeneous antibody populations carrying variable numbers of DNA docking strands, compromising quantitative reliability. Here, we introduce site-selective disulfide rebridging using pyridazinedione chemistry to generate stoichiometrically defined antibody-DNA conjugates with either one (OAR1) or four (OAR4) DNA-PAINT docking sequences per antibody. Using nuclear pore complexes as a benchmark, we demonstrate that both OAR1 and OAR4 probes yield narrow qPAINT index distributions that exhibit the expected proportional dependence on protein copy number, enabling reliable discrimination between singly and doubly labeled Nup96 dimers. In contrast, conventional lysine-based conjugation produces broadened distributions and systematic overestimation due to uncontrolled DNA loading. These results establish selective antibody-DNA conjugation as a prerequisite for accurate quantitative DNA-PAINT in complex cellular environments.