Homogeneous Antibody-DNA Conjugates Using Unmodified Oligonucleotides and Photo-Cross-Linkable Protein G-HUH...

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-stranded 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 Mg²⁺ compared to the more commonly used Mn²⁺ cofactor ion. Although formation of the phosphotyrosine 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-cross-linking 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 approach to prepare homogeneous antibody-DNA conjugates with 1:1 stoichiometry.