Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy
Philipp Werther (University of Heidelberg)
Klaus Yserentant (University of Heidelberg)
Felix Braun (University of Heidelberg)
Kristin Grußmayer (École Polytechnique Fédérale de Lausanne, TU Delft - BN/Afdelingsbureau, TU Delft - BN/Kristin Grussmayer Lab, Kavli institute of nanoscience Delft)
Vytautas Navikas (École Polytechnique Fédérale de Lausanne)
Miao Yu (Johannes Gutenberg-University Mainz)
Zhibin Zhang (University of Heidelberg, Harbin Institute of Technology)
Michael J. Ziegler (Max Planck Institute for Medical Research, University of Heidelberg)
Christoph Mayer (University of Heidelberg)
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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.
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