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

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, while 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 encoding the green fluorescent protein iLOV from ten functionalized oligonucleotides containing 5'-azide and 3'-alkyne units. The resulting click-linked iLOV contains eight triazoles at the sites of chemical ligation, yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in E. coli. We demonstrate the power and potential of our one-pot gene assembly method by preparing an epigenetically modified variant of the iLOV gene.

More information

Identifiers

ORCID for Ali Tavassoli: orcid.org/0000-0002-7420-5063

Catalogue record

Export record

Altmetrics