Reduced Non-Specific Binding of Super-Resolution DNA-PAINT Markers by Shielded DNA-PAINT Labeling Protocols.
DNA‐PAINT
immunostaining
single molecule
super‐resolution
Journal
Small (Weinheim an der Bergstrasse, Germany)
ISSN: 1613-6829
Titre abrégé: Small
Pays: Germany
ID NLM: 101235338
Informations de publication
Date de publication:
18 Oct 2024
18 Oct 2024
Historique:
revised:
09
09
2024
received:
19
06
2024
medline:
18
10
2024
pubmed:
18
10
2024
entrez:
18
10
2024
Statut:
aheadofprint
Résumé
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.
Identifiants
pubmed: 39422065
doi: 10.1002/smll.202405032
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2405032Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/T007176/1
Pays : United Kingdom
Organisme : Bio-Techne and the University of Exeter
Informations de copyright
© 2024 The Author(s). Small published by Wiley‐VCH GmbH.
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