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

Jonathan SchwachKsenia KolobyninaKatharina BrandstetterMarcus GerlachPhilipp OchtropJonas HelmaChristian P R HackenbergerHartmann HarzM Cristina CardosoHeinrich LeonhardtAndreas Stengl
Published in: Chembiochem : a European journal of chemical biology (2020)
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.
Keyphrases
  • high resolution
  • single molecule
  • cancer therapy
  • nucleic acid
  • binding protein
  • optical coherence tomography
  • drug delivery
  • amino acid
  • fluorescence imaging