Site‐Specific Antibody Fragment Conjugates for Reversible Staining in Fluorescence Microscopy
| Autor: | Ksenia Kolobynina, Christian P. R. Hackenberger, Heinrich Leonhardt, Jonathan Schwach, Katharina Brandstetter, Hartmann Harz, Philipp Ochtrop, M. Cristina Cardoso, Marcus Gerlach, Andreas Stengl, Jonas Helma |
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| Rok vydání: | 2020 |
| Předmět: |
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 |
| Zdroj: | Chembiochem |
| ISSN: | 1439-7633 1439-4227 |
| DOI: | 10.1002/cbic.202000727 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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