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Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation

Nature Chemistry volume 9, pages 1089–1098 (2017)Cite this article

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Abstract

The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5ʹ-azide and 3ʹ-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

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Figure 1: One-pot gene synthesis by click-DNA ligation.
Figure 2: Synthesis of alkyne-modified dC and loading onto a solid support in preparation for oligonucleotide synthesis.
Figure 3: Characterization of the click-linked iLOV gene.
Figure 4: Assembly of the click-ligated iLOV plasmid.
Figure 5: Biocompatibility of click-linked iLOV in E. coli cells.
Figure 6: Click-assembly and characterization of epigenetically modified iLOV.

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Acknowledgements

We thank R. Tsien for his kind gift of pRSET-mCherry, N.J. Wright for assistance with CD spectroscopy, and D. Brown for access to ATDBio's oligonucleotide synthesis facilities. This work was supported by UK BBSRC grants BB/J001694/2 and BB/M025624/1.

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Authors and Affiliations

  1. Chemistry, University of Southampton, Southampton, SO17 1BJ, UK

    Mikiembo Kukwikila & Ali Tavassoli

  2. ATDBio, Chemistry, University of Southampton, Southampton, SO17 1BJ, UK

    Nittaya Gale

  3. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, OX1 3TA, United Kingdom

    Afaf H. El-Sagheer & Tom Brown

  4. Department of Science and Mathematics, Chemistry Branch, Suez University, Suez, 43721, Egypt

    Afaf H. El-Sagheer

  5. Institute for Life Science, University of Southampton, Southampton, SO17 1BJ, UK

    Ali Tavassoli

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Contributions

A.T. conceived, designed and supervised the study. M.K. synthesized the modified DNA bases, conducted the click-DNA assembly, carried out all the biological experiments and analysed data. N.G. and A.H.E.-S. conducted the oligonucleotide synthesis, mass spectrometry and analysed the data. T.B. provided material and supervision for oligonucleotide synthesis. The manuscript was written by M.K. and A.T. with input from all the authors.

Corresponding authors

Correspondence to Tom Brown or Ali Tavassoli.

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Competing interests

M.K. and A.T. are co-inventors on the US patent application ‘one-pot gene synthesis’. A.H.E.S. and T.B. are co-inventors on US patent 8,846,883 B2 Oligonucleotide Ligation.

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Kukwikila, M., Gale, N., El-Sagheer, A. et al. Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation. Nature Chem 9, 1089–1098 (2017). https://doi.org/10.1038/nchem.2850

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