Schwach, J., Kolobynina, K., Brandstetter, K., Gerlach, M., Ochtrop, P., Helma, J., Hackenberger, C.P.R., Harz, H., Cardoso, M.C., Leonhardt, H., and Stengl, A.
(IMPRS-LS students are in bold)
Chembiochem, 2020, [Epub ahead of print].
Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy
Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences, which 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 PNAs, allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub-tag Ⓡ 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.