Reduced Non‐Specific Binding of Super‐Resolution DNA‐PAINT Markers by Shielded DNA‐PAINT Labeling Protocols

Abstract The DNA‐based single molecule super‐resolution imaging approach, DNA‐PAINT, can achieve nanometer resolution of single targets. However, the approach can suffer from significant non‐specific background signals originating from non‐specifically bound DNA‐conjugated DNA‐PAINT secondary antibodies as shown here. Using dye‐modified oligonucleotides the location of DNA‐PAINT secondary antibody probes can easily be observed with widefield imaging prior to beginning a super‐resolution measurement. This reveals that a substantial proportion of DNA probes can accumulate, non‐specifically, within the nucleus, as well as across the cytoplasm, of cells. Here, Shielded DNA‐PAINT labeling is introduced, a method using partially or fully double‐stranded docking strand sequences, prior to labeling, in buffers with increased ionic strength to greatly reduce non‐specific interactions in the nucleus as well as the cytoplasm. This new labeling approach is evaluated against various conditions and it is shown that applying Shielded DNA‐PAINT can reduce non‐specific events approximately five‐fold within the nucleus. This marked reduction in non‐specific binding of probes during the labeling procedure is comparable to results obtained with unnatural left‐handed DNA albeit at a fraction of the cost. Shielded DNA‐PAINT is a straightforward adaption of current DNA‐PAINT protocols and enables nanometer precision imaging of nuclear targets with low non‐specific backgrounds.