Homogeneous antibody-DNA-conjugates using unmodified oligonucleotides and photo-crosslinkable protein G-HUH endonuclease fusion proteins

Antibody-DNA conjugates are increasingly used in analytical biochemistry and nanotechnology. However, current methods for their preparation often lack specificity, resulting in heterogeneous products. Here, we present a novel strategy to covalently label the Fc domain of antibodies with single-strand DNA in a site-specific and stoichiometrically controlled manner. This method employs unmodified oligonucleotides and a fusion protein consisting of a protein G dimer linked to an HUH endonuclease. We first evaluated the sequence specificity and optimal conditions for DNA attachment to three HUH endonuclease variants, DCV, PCV2, and WDV. Our results show increased sequence specificity in the presence of Mg ²⁺ compared to the more commonly used Mn ²⁺ cofactor ion. Although formation of the phospho-tyrosine bond is found to be reversible, no significant hydrolysis of the protein-DNA conjugates is observed for up to 8 days at room temperature. The DCV domain allowed essentially complete formation of DNA-fusion protein conjugates at a 1:1 protein-DNA ratio, eliminating the need for removal of excess oligos. Subsequent photo-crosslinking yielded antibody-DNA conjugates that were successfully used in a proximity extension assay (PEA) to detect TNFα and IL-6. Our method provides an efficient, low cost and generally applicable method to prepare homogeneous antibody–DNA conjugates with 1:1 stoichiometry.