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

Mikiembo Kukwikila, Nittaya Gale, Afaf H. El-Sagheer, Tom Brown, Ali Tavassoli

Research output: Contribution to journalArticlepeer-review

70 Citations (Scopus)

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 5'-azide and 3'-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.

Original languageEnglish
Pages (from-to)1089-1098
Number of pages10
JournalNature Chemistry
Volume9
DOIs
Publication statusPublished - Nov 2017
Externally publishedYes

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