Impact of Docking Strand Design on Spatial Resolution in DNA‐Points Accumulation for Imaging in Nanoscale Topography

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) has become a widely adopted single-molecule localization microscopy (SMLM) technique owing to its high spatial resolution, versatile labeling strategies, and theoretically unlimited multiplexing capability. Recent developments in repetitive docking strand designs have enabled faster image acquisition by increasing the number of potential binding motifs per target. However, the effect of such architectural modifications on effective spatial resolution remains largely unexplored. Here, we systematically quantify how repetitive docking strands influence localization distributions and effective resolution using the well-defined geometry of the trimeric proliferating cell nuclear antigen (PCNA) as a model system. Whereas classical single-motif docking strands resolve the expected ∼6 nm spacing between PCNA subunits with high precision, repetitive docking motifs produce broadened localization distributions, despite comparable localization precision. Our results suggest that spatial blurring arises from a combination of variable binding site geometry, rotational flexibility of elongated multivalent DNA docking sequences, as well as the dynamic behavior of imager strands. This study provides a quantitative framework for understanding how docking strand architecture determines resolution limits in DNA-PAINT and underscores the need to balance multiplexing and imaging speed with structural fidelity. Our results thus offer guidance for the rational design of docking strands for high-precision DNA-PAINT imaging of protein complexes.