HUH-Enabled Programmable Antibody-DNA Conjugates for Profiling Receptor- Specific Cellular Mechanics

Abstract

Cell-surface receptors integrate biochemical identity with mechanical information, yet methods for measuring receptor-specific force transmission across cell populations remain limited by the difficulty of coupling diverse recognition reagents to nucleic-acid tension probes. Here, we establish HUH endonuclease chemistry as a modular interface between antibodies and DNA- based mechanosensors. We develop two complementary strategies: genetically encoded HUH- antibody fusions that generate site-defined antibody-oligonucleotide conjugates in a single reaction, and a photocrosslinkable Protein G-HUH adaptor that enables covalent attachment of existing antibodies to DNA probes. Both approaches preserve antibody recognition while providing a programmable nucleic-acid handle for Rupture and Deliver Tension Gauge Tethers (RAD-TGTs). Using antibodies against beta1 integrin and HER2, we resolve receptor-specific mechanical phenotypes across cancer cell lines. Combining orthogonal features of beta1-integrin engagement and HER2 mechanical heterogeneity provides greater discrimination among cell types than either measurement alone, demonstrating that multidimensional mechanical phenotypes contain information not captured by individual force measurements. We further extend the platform to DNA:PNA tension probes to mitigate extracellular nuclease degradation and use antibody- functionalized RAD-TGTs to quantify force-dependent receptor engagement and pharmacological responses in immune cells. Together, these studies establish a modular antibody-to-nucleic-acid interface that expands DNA-based tension sensing beyond a restricted set of ligands and enables quantitative, multidimensional profiling of receptor-specific mechanical behavior across heterogeneous cell populations.