Programmable Antibody-DNA Conjugation via HUH-Tags Enables Quantitative Measurement of Receptor-Specific Adhesion Dynamics

Abstract

Antibody-DNA oligonucleotide conjugates (AOCs) are widely used for molecular assembly and cellular analysis, yet current approaches for generating these conjugates often rely on nonspecific chemistries that produce heterogeneous products. Here, we present two complementary strategies for generating site-specific AOCs using covalent DNA-linking HUH endonucleases. In one approach, recombinant antibodies are genetically fused to HUH-tags to enable direct, site-specific DNA conjugation. In the second, off-the-shelf antibodies are indirectly linked to HUH-tags using a photocrosslinkable Protein G-HUH fusion, enabling covalent Fc-directed attachment. Both strategies yield homogeneous AOCs while preserving antigen binding affinity. We apply these conjugates to a DNA-based mechanochemical assay, termed rupture-and-deliver tension gauge tethers (RAD-TGTs), which converts receptor-mediated adhesion forces into intracellular delivery of a fluorescent oligonucleotide payload. By tuning duplex stability, we define adhesion dynamics across multiple mechanical regimes. Using HER2- and beta1-integrin-targeting AOCs, we identify receptor-specific adhesion signatures and uncover cooperative interactions between receptor systems in a panel of cancer cell lines. Dual-color probes enable multiplexed single-cell mechanical phenotyping, and application to primary NK cells reveals dose-dependent responses to integrin modulators. These results establish a generalizable platform for site-defined AOC generation and for quantitative, high-throughput measurement of receptor-mediated adhesion dynamics.