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Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.

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 : a European journal of chemical biology, ISSN 1439-7633,
DOI: 10.1002/cbic.202000727,
[Article]

Abstract

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.

Item Type: Article
Erschienen: 2020
Creators: 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
Title: Site-Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy.
Language: English
Abstract:

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.

Journal or Publication Title: Chembiochem : a European journal of chemical biology
Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 24 Nov 2020 07:17
DOI: 10.1002/cbic.202000727
Identification Number: pmid:33207032
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