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

Journal article

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.

Authors: Kukwikila, M; Gale, N; El-Sagheer, A; Brown, T; Tavassoli, A

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Publishing Group
• Journal: Nature Chemistry
• Volume: 9
• Pages: 1089-1098
• Publication date: 2017-09-11
• Acceptance date: 2017-07-25
• DOI: 10.1038/NCHEM.2850
• EISSN: 1755-4349
• ISSN: 1755-4330
• Keywords: DNA; Nucleic acids; Synthetic biology