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bioRxiv · 10.64898/2026.11.11.765849

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

Ehrl, A.·Ziska, E.·Shen, K.·Morrissey, M. A.·Douglas, S. M.·Taylor, M. J.
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Abstract

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.

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Ehrl, A., Ziska, E., Shen, K., Morrissey, M. A., Douglas, S. M., Taylor, M. J.. 2026-11-13. HUH-tag conjugation enables accessible, site-specific functionalization of DNA origami with signaling-competent proteins. https://doi.org/10.64898/2026.11.11.765849

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