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  Reversible live-cell labeling with retro-engineered HaloTags enables long-term high- and super-resolution imaging

Holtmannspötter, M., Wienbeuker, E., Dellmann, T., Watrinet, I., Garcia-Sáez, A. J., Johnsson, K., et al. (2023). Reversible live-cell labeling with retro-engineered HaloTags enables long-term high- and super-resolution imaging. Angewandte Chemie International Edition, 62(18): e202219050, pp. 1-8. doi:10.1002/anie.202219050.

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Item Permalink: https://hdl.handle.net/21.11116/0000-000C-EA67-9 Version Permalink: https://hdl.handle.net/21.11116/0000-000D-06DA-7
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Creators

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 Creators:
Holtmannspötter, Michael, Author
Wienbeuker, Eike, Author
Dellmann, Timo, Author
Watrinet, Isabelle, Author
Garcia-Sáez, Ana J, Author
Johnsson, Kai1, Author           
Kurre, Rainer, Author
Piehler, Jacob, Author
Affiliations:
1Chemical Biology, Max Planck Institute for Medical Research, Max Planck Society, ou_2364732              

Content

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Free keywords: Fluorescent Probes; Protein Engineering; STED Microscopy; Self-Labeling Enzyme; Single Molecule Tracking
 Abstract: Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006 s-1 ) and reHaloTagF (≈0.055 s-1 ). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.

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Language(s): eng - English
 Dates: 2022-12-232023-02-032023-02-032023-04-24
 Publication Status: Issued
 Pages: 8
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: URI: https://pubmed.ncbi.nlm.nih.gov/36735334/
DOI: 10.1002/anie.202219050
 Degree: -

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Title: Angewandte Chemie International Edition
  Abbreviation : Angew. Chem., Int. Ed.
Source Genre: Journal
 Creator(s):
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Publ. Info: Weinheim : Wiley-VCH
Pages: - Volume / Issue: 62 (18) Sequence Number: e202219050 Start / End Page: 1 - 8 Identifier: ISSN: 1433-7851
CoNE: https://pure.mpg.de/cone/journals/resource/1433-7851