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.

中文翻译：

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一锅式单击-DNA连接可组装生物相容性三唑连接的基因

寡核苷酸的化学合成及其酶介导的组装成基因和基因组的方法已大大提高了多种科学学科的水平。但是，这些方法并非没有缺点。寡核苷酸的酶促扩增和连接到基因和基因组中使自动化具有挑战性，并且将表观遗传信息和/或修饰的碱基特异性结合到大型构建体中是不可行的。在这里，我们介绍一种通过单击DNA连接将寡核苷酸组装成基因的全化学一锅法。我们从包含5′-叠氮化物和3′-炔基单元的十个功能化寡核苷酸合成了编码绿色荧光蛋白iLOV的335个碱基对基因。产生点击链接的iLOV该基因在化学连接位点包含八个三唑，但具有完全的生物相容性；它在体外由DNA聚合酶复制，并在大肠杆菌中编码功能性iLOV蛋白。我们通过准备一个表观遗传修饰的iLOV基因变体来证明我们的一锅法基因组装方法的力量和潜力。