3 years ago

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

Assembly of a biocompatible triazole-linked gene by one-pot click-DNA ligation
Ali Tavassoli, Mikiembo Kukwikila, Nittaya Gale, Afaf H. El-Sagheer, Tom Brown
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.

Publisher URL: https://www.nature.com/articles/nchem.2850

DOI: 10.1038/nchem.2850

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