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Schwach, Jonathan; Kolobynina, Ksenia; Brandstetter, Katharina; Gerlach, Marcus; Ochtrop, Philipp; Helma, Jonas; Hackenberger, Christian P. R.; Harz, Hartmann; Cardoso, M. Cristina; Leonhardt, Heinrich; Stengl, Andreas (2020): Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy. In: Chembiochem, Vol. 22, No. 7: pp. 1205-1209
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Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site-specific modification of proteins. Antibody-oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence-encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag (R) technology in combination with Cu-catalyzed azide-alkyne cycloaddition for the site-specific conjugation of single DNA and PNA strands to an eGFP-binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence-specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP-tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody-oligonucleotides for reversible immunofluorescence imaging.