Article

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

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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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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.

Author information

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. Chemistry Branch, Department of Science and Mathematics, 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.

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.

Corresponding authors

Correspondence to Tom Brown or Ali Tavassoli.

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