HUH-tag conjugation enables accessible, site-specific functionalization of DNA origami with signaling-competent proteins

DNA origami enables precise nanometer scale control over the number, spacing, and geometry of biomolecules, making it a powerful tool for dissecting spatially organized biological processes such as receptor signaling. However, functionalizing origami with proteins remains a bottleneck restricting the technology to specialized laboratories. Here we show that viral-derived HUH endonuclease tags, small (~10 kDa) domains that form a covalent phosphotyrosine bond with short unmodified ssDNA, provide a simple and cost-effective route to decorating DNA origami with functional proteins. Using interleukin-1{beta} (IL-1{beta}) as a model ligand, we generated recombinant IL-1{beta}-HUH, achieved efficient site-specific conjugation to unmodified ssDNA within minutes, and coupled the conjugates to a DNA origami pegboard with tunable stoichiometry. IL-1{beta}-functionalized origami triggered ligand-dependent signalling in living cells and allowed visualization of the recruitment of downstream signaling molecules, confirming that HUH-tag conjugation preserves functionality. Because this strategy requires only standard molecular biology equipment and unmodified DNA, it lowers a technical and financial barrier to DNA origami-based experimentation, opening the approach to a broader range of biological questions and laboratories outside the DNA nanotechnology field.