Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody–DNA Conjugates
journal contributionposted on 25.10.2019, 16:33 by Christiane Stiller, Hooman Aghelpasand, Tobias Frick, Kristina Westerlund, Afshin Ahmadian, Amelie Eriksson Karlström
Antibody–DNA conjugates are powerful tools for DNA-assisted protein analysis. Growing usage of these methods demands efficient production of high-quality conjugates. We developed an easy and fast synthesis route yielding covalent antibody–DNA conjugates with a defined conjugation site and low batch-to-batch variability. We utilize the Z domain from protein A, containing the unnatural amino acid 4-benzoylphenylalanine (BPA) for photoaffinity labeling of the antibodies’ Fc region. Z(xBPA) domains are C-terminally modified with triple-glycine (G3)-modified DNA-oligonucleotides via enzymatic Sortase A coupling. We show reliable modification of the most commonly used IgG’s. To prove our conjugates’ functionality, we detected antibody–antigen binding events in an assay called Droplet Barcode Sequencing for Protein analysis (DBS-Pro). It confirms not only retained functionality for both conjugate parts but also the potential of using DBS-Pro for quantifying protein abundances. As intermediates are easily storable and our approach is modular, it offers a convenient strategy for screening various antibody–DNA conjugates using the same starting material.