Fast and Efficient Fc-Specific Photoaffinity Labeling To Produce Antibody-DNA Conjugates.

Christiane Stiller,Tobias Frick,Kristina Westerlund,Amelie Eriksson Karlström,Afshin Ahmadian,Hooman Aghelpasand

doi:10.1021/acs.bioconjchem.9b00548

Christiane Stiller, Tobias Frick + Show 4 more

Open Access

https://doi.org/10.1021/acs.bioconjchem.9b00548

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Abstract

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.

Highlights

Since the first report on immuno-polymerase chain reaction (PCR) in 1992, antibody−DNA conjugates have been recognized as a powerful tool for analyses of biomolecules.[1]
After this initial study major improvements as well as various other DNA-assisted methods, such as proximity ligation and extension assays (PLA/PEA), rolling circle amplification (RCA), and barcoded proteomics employing antibody−DNA conjugates, have been developed.[2−9] The growing usage of these methods for, e.g., biomarker discovery or pathogen detection leads to a high demand for reproducible, fast, and high-yielding conjugation methods allowing researchers to focus on the final assay instead of antibody−DNA conjugate production
DNA conjugates consists of three subsequent steps, namely, (1) Escherichia coli expression of Z domains carrying the unnatural amino acid BPA introduced using Amber suppression and a C-terminal Sortase A recognition motif, (2) the Sortase A-catalyzed modification of isolated Z(xBPA) domains with G3-modified DNA-oligonucleotides, and (3) the Fc region specific photoaffinity labeling of antibodies with Z(xBPA)−DNA conjugates (Figure 1)

Summary

Introduction

Since the first report on immuno-PCR in 1992, antibody−DNA conjugates have been recognized as a powerful tool for analyses of biomolecules.[1]. If the targeted antibody requires amine-containing buffer supplements for stabilization, such as commonly used BSA, these are colabeled, increasing the consumption of DNA and complicating purification even further. To overcome these obstacles, various methods for the site-specific labeling of antibodies with DNA have been developed, which can be distinguished in two major groups: the first one employs genetic modification of the targeted antibody, thereby installing a bioorthogonal reaction site on it.[14−16] These approaches, come with long development times for antibody-producing cell lines and are most useful when high amounts of antibody conjugates and continuous supply are needed. In combination with these domains, e.g., the Tus-Ter-lock or the ΦX174 geneA* system were used to mediate DNA binding.[19,20] these

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