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      Green- to far-red-emitting fluorogenic tetrazine probes – synthetic access and no-wash protein imaging inside living cells†

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          Abstract

          Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging.

          Abstract

          Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging. 1,2,4,5-Tetrazines are known substrates for the bioorthogonal inverse electron demand Diels–Alder reaction (DA inv) and tetrazine substituted fluorophores can exhibit fluorogenic properties. Herein, we report the synthesis of a palette of novel fluorogenic tetrazine dyes derived from widely-used fluorophores that cover the entire emission range from green to far-red. We demonstrate the power of the new fluorogenic probes in fixed and live cell labeling experiments and present the first example of intracellular live cell protein imaging using tetrazine-based probes under no-wash conditions.

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          A general method for the covalent labeling of fusion proteins with small molecules in vivo.

          Antje Keppler, Susanne Gendreizig, Thomas Gronemeyer … (2003)
          Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.
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            HaloTag: a novel protein labeling technology for cell imaging and protein analysis.

            Georgyi V Los, Lance P Encell, Mark G McDougall … (2008)
            We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-kappaB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of protein--protein and protein--DNA complexes.
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              Fluorescent probes for super-resolution imaging in living cells.

              Marta Fernandez-Suarez, Alice Ting (2008)
              In 1873, Ernst Abbe discovered that features closer than approximately 200 nm cannot be resolved by lens-based light microscopy. In recent years, however, several new far-field super-resolution imaging techniques have broken this diffraction limit, producing, for example, video-rate movies of synaptic vesicles in living neurons with 62 nm spatial resolution. Current research is focused on further improving spatial resolution in an effort to reach the goal of video-rate imaging of live cells with molecular (1-5 nm) resolution. Here, we describe the contributions of fluorescent probes to far-field super-resolution imaging, focusing on fluorescent proteins and organic small-molecule fluorophores. We describe the features of existing super-resolution fluorophores and highlight areas of importance for future research and development.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Chem Sci
                Journal ID (iso-abbrev): Chem Sci
                Title: Chemical Science
                Publisher: Royal Society of Chemistry
                ISSN (Print): 2041-6520
                ISSN (Electronic): 2041-6539
                Publication date (Print): 1 February 2017
                Publication date (Electronic): 21 October 2016
                Volume: 8
                Issue: 2
                Pages: 1506-1510
                Affiliations
                [a ] Institut für Pharmazie und Molekulare Biotechnologie , Ruprecht-Karls-Universität Heidelberg , Im Neuenheimer Feld 364 , 69120 Heidelberg , Germany . Email: wombacher@ 123456uni-heidelberg.de
                Author notes

                ‡These authors contributed equally.

                Article
                Publisher ID: c6sc03879d
                DOI: 10.1039/c6sc03879d
                PMC ID: 5452268
                PubMed ID: 28572909
                SO-VID: 36594ac0-7ff2-4b6a-a26b-e6ad03be7ad4
                Copyright © This journal is © The Royal Society of Chemistry 2016
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported License ( http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 30 August 2016
                Date accepted : 19 October 2016
                Categories
                Subject: Chemistry

                Notes

                †Electronic supplementary information (ESI) available: Synthetic procedures and spectroscopic data, details of cell experiments and imaging. See DOI: 10.1039/c6sc03879d


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