Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells

Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intrac...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2021-09, Vol.60 (40), p.22075-22080
Main Authors: Schneider, Anselm F. L., Benz, Laila S., Lehmann, Martin, Hackenberger, Christian P. R.
Format: Article
Language:English
Subjects:
Cell division
cell-penetrating peptides
Cells (biology)
Deoxyribonucleic acid
DNA
DNA biosynthesis
DNA probes
DNA repair
Fluorescence
Functional analysis
Genetic code
Intracellular
Lamins
Low concentrations
Microscopy
Nanobodies
nanobody
Permeability
Proliferating cell nuclear antigen
Proteins
Replication
semi-synthesis
STED
super-resolution microscopy
Vitamin E
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Summary:Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis. Cell‐permeable nanobodies equipped with a fluorophore and a photostabilizer are introduced as powerful tools for live‐cell super‐resolution microscopy. By using intramolecular photostabilization dynamic processes can be monitored in untransfected living cells.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.202103068