1. Site‐Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy
- Author
-
Ksenia Kolobynina, Christian P. R. Hackenberger, Heinrich Leonhardt, Jonathan Schwach, Katharina Brandstetter, Hartmann Harz, Philipp Ochtrop, M. Cristina Cardoso, Marcus Gerlach, Andreas Stengl, and Jonas Helma
- Subjects
Peptide Nucleic Acids ,Azides ,Immunoconjugates ,Green Fluorescent Proteins ,Peptide ,immunofluorescence staining ,Tub-tag labelling ,Immunofluorescence ,Biochemistry ,Catalysis ,Cell Line ,law.invention ,chemistry.chemical_compound ,law ,super-resolution microscopy ,ddc:572 ,572 Biochemie ,medicine ,Fluorescence microscope ,Humans ,Immunoglobulin Fragments ,Molecular Biology ,chemistry.chemical_classification ,peptide nucleic acids ,Cycloaddition Reaction ,medicine.diagnostic_test ,Oligonucleotide ,Communication ,Organic Chemistry ,DNA ,Single-Domain Antibodies ,Communications ,nanobodies ,540 Chemie und zugeordnete Wissenschaften ,Microscopy, Fluorescence ,chemistry ,Alkynes ,ddc:540 ,antibody conjugates ,Nucleic acid ,Biophysics ,Recombinant DNA ,Molecular Medicine ,Copper ,Conjugate - Abstract
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., Protein‐oligonucleotide conjugates. We present a novel strategy for the efficient and site‐specific generation of nanobody‐oligonucleotide conjugates in a 1 : 1 stoichiometry by Tub‐tag‐mediated conjugation. We show a proof‐of‐concept that these conjugates can readily be used for reversible staining in confocal fluorescence microscopy.
- Published
- 2020
- Full Text
- View/download PDF