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Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

Nature Chemistry volume 6, pages 804–809 (2014)Cite this article

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Abstract

DNA–protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA–protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

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Figure 1: Expanding the concept of DTS for site-selective conjugation of DNA to proteins.
Figure 2: Site-selective DNA-templated coupling of an activated ester to His6-tagged GFP.
Figure 3: DTPC labelling of Tf and cellular uptake of the conjugate.
Figure 4: Targeting the Fc domain histidine cluster of IgG1 antibodies.

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Acknowledgements

The work was supported financially by the Danish National Research Foundation (grant number DNRF81) and Aarhus University, Faculty of Science and Technology. The authors are grateful to J. B. Knudsen for assistance in the making of the manuscript figures. The authors thank K. Stødkilde-Jørgensen for providing His6-tagged endoglycosidase H protein.

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Author notes

  1. Christian B. Rosen and Anne L. B. Kodal: These authors contributed equally to this work

Authors and Affiliations

  1. Center for DNA Nanotechnology and Interdisciplinary Nanoscience Center, Gustav Wieds Vej 14, Aarhus C, 8000, Denmark

    Christian B. Rosen, Anne L. B. Kodal, Jesper S. Nielsen, David H. Schaffert, Anders H. Okholm, Niels V. Voigt, Jørgen Kjems, Thomas Tørring & Kurt V. Gothelf

  2. Department of Chemistry, Langelandsgade 140, Aarhus C, 8000, Denmark

    Christian B. Rosen, Anne L. B. Kodal, Niels V. Voigt, Thomas Tørring & Kurt V. Gothelf

  3. Department of Molecular Biology and Genetics, Aarhus University, C. F. Møllers Allé 3, 8000 Aarhus C, Denmark

    Jesper S. Nielsen, David H. Schaffert, Anders H. Okholm & Jørgen Kjems

  4. Department of Molecular Biology and Genetics, Science Park, Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark

    Carsten Scavenius & Jan J. Enghild

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Contributions

C.B.R. and A.L.B.K. contributed equally to this work. C.B.R., A.L.B.K., T.T. and K.V.G. planned the research and wrote the paper. C.B.R, A.L.B.K. and T.T. developed the method, performed experiments and analysed the data. J.S.N. performed the Tf-receptor binding studies and assisted in the gel blotting. D.H.S. and A.H.O. conducted the cell studies. A.H.O. assisted in the enzymatic assay experiments. C.S. performed the MS/MS experiments. T.T., N.V.V. and K.V.G. conceived the study. J.J.E., J.K. and K.V.G. supervised the project. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to Thomas Tørring or Kurt V. Gothelf.

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Competing interests

C.B.R., A.L.B.K., T.T. and K.V.G. have filed a provisional patent application regarding the current work. The other authors declare no competing financial interests.

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Rosen, C., Kodal, A., Nielsen, J. et al. Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins. Nature Chem 6, 804–809 (2014). https://doi.org/10.1038/nchem.2003

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