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

## TL;DR

This study explores how the design of DNA docking strands affects the spatial resolution in DNA-PAINT imaging, revealing that repetitive designs can blur details despite high precision.

## Contribution

The paper introduces a quantitative framework linking docking strand architecture to resolution limits in DNA-PAINT imaging.

## Key findings

- Repetitive docking motifs lead to broadened localization distributions despite high localization precision.
- Spatial blurring is caused by variable binding site geometry and DNA strand flexibility.
- The study provides guidance for designing docking strands to balance speed and structural fidelity.

## Abstract

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.

The design of DNA‐PAINT docking strands critically influences spatial resolution and signal stability. By comparing single (1 × R1) and repetitive (5 × R1) docking architectures on PCNA, we reveal how binding kinetics and localization precision determine the accuracy of sub‐10 nm imaging in protein nanostructures.© 2026 WILEY‐VCH GmbH