Bio-orthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy

Philipp Werther, Klaus Yserentant, Felix Braun, Kristin Grußmayer, Vytautas Navikas, Miao Yu, Zhibin Zhang, Michael J. Ziegler, Christoph Mayer, Antoni J. Gralak, Marvin Busch, Weijie Chi, Frank Rominger, Aleksandra Radenovic, Xiaogang Liu, Edward A. Lemke, Tiago Buckup, Dirk Peter Herten, Richard Wombacher*

*Corresponding author for this work

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Abstract

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

Original languageEnglish
Pages (from-to)1561–1571
Number of pages11
JournalACS Central Science
Volume7
Issue number9
Early online date20 Aug 2021
DOIs
Publication statusPublished - 22 Sept 2021

Bibliographical note

Funding Information:
RW and EAL acknowledge funding from the Deutsche Forschungsgemeinschaft DFG (SPP1623) and DPH from the Federal Ministry of Education and Research (BMBF/VDI; MorphiQuant3D and Switch-Click-Microscopy) and the DFG (HE4559/5-1, HE4559/6-1). DPH gratefully acknowledges funding by the Centre of Membrane Proteins and Receptors (COMPARE, Universities of Birmingham and Nottingham). EAL also thanks ERC SMPFv2.0 for funding. AR, KG, and VN thank the EPFL BioImaging & Optics Core Facility (EPFL-BIOP) and Horizon 2020 research and innovation program of the European Union via grant 686271/SEFRI 16.0047 for funding. XL acknowledges funding from Singapore University of Technology and Design (T1SRCI17126). ZZ acknowledges funding from the National Natural Science Foundation of China (Grant No. 61675057) and China Scholarship Council. MJZ acknowledges a fellowship by the Carl-Zeiss-Stiftung. We thank Carina Lämmle for providing a tetrazine compound for Figure S22 . We gratefully acknowledge access to the Nikon Imaging Center at Heidelberg University. We thank Daniel Wolf and Frederik Weber for experimental help and Prof. Dr. Andres Jäschke for constant support.

Publisher Copyright:
© 2021 The Authors. Published by American Chemical Society.

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)

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