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Site‐Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy
Author(s) -
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
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000727
Subject(s) - oligonucleotide , conjugate , chemistry , fluorescence microscope , peptide , recombinant dna , nucleic acid , dna , microbiology and biotechnology , immunofluorescence , fluorescence , antibody , biochemistry , combinatorial chemistry , biology , mathematical analysis , physics , mathematics , quantum mechanics , immunology , gene
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® 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.