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

Autor: Lučinskaitė E; Department of Physiology, University of Bern, Bern, 3012, Switzerland., Bokhobza AFE; Department of Physiology, University of Bern, Bern, 3012, Switzerland., Stannard A; Department of Chemistry, Imperial College London, London, W12 OBZ, UK., Meletiou A; Department of Physiology, University of Bern, Bern, 3012, Switzerland., Estell C; Living Systems Institute, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK., West S; Living Systems Institute, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK., Michele LD; Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB3 0AS, UK., Soeller C; Department of Physiology, University of Bern, Bern, 3012, Switzerland., Clowsley AH; Department of Physiology, University of Bern, Bern, 3012, Switzerland.
Jazyk: angličtina
Zdroj: Small (Weinheim an der Bergstrasse, Germany) [Small] 2024 Oct 18, pp. e2405032. Date of Electronic Publication: 2024 Oct 18.
DOI: 10.1002/smll.202405032
Abstrakt: 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.
(© 2024 The Author(s). Small published by Wiley‐VCH GmbH.)
Databáze: MEDLINE
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