Bai Jingsi, Zou Jinrong, Cao Yijun, Du Yuhui, Chen Tingjian
MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, P. R. China.
ACS Synth Biol. 2023 Sep 15;12(9):2676-2690. doi: 10.1021/acssynbio.3c00250. Epub 2023 Aug 17.
Unnatural base pairs (UBPs) have been developed to expand the genetic alphabet and . UBP dNaM-dTPT3 and its analogues have been successfully used to construct the first set of semi-synthetic organisms, which suggested the great potential of UBPs to be used for producing novel synthetic biological parts. Two prerequisites for doing so are the facile manipulation of DNA containing UBPs with common tool enzymes, including DNA polymerases and ligases, and the easy availability of UBP-containing DNA strands. Besides, for the application of UBPs in phage synthetic biology, the recognition of UBPs by phage enzymes is essential. Here, we first explore the recognition of dNaM-dTPT3 by a family B DNA polymerase from bacteriophage, T4 DNA polymerase D219A. Results from primer extension, steady-state kinetics, and gap-filling experiments suggest that T4 DNA polymerase D219A can efficiently and faithfully replicate dNaM-dTPT3, and efficiently fill a gap by inserting dTPT3TP or its analogues opposite dNaM. We then systematically explore the recognition of dNaM-dTPT3 and its analogues by different DNA ligases from bacteriophages and find that these DNA ligases are generally able to efficiently ligate the DNA nick next to dNaM-dTPT3 or its analogues, albeit with slightly different efficiencies. These results suggest more enzymatic tools for the manipulation of dNaM-dTPT3 and indicate the potential use of dNaM-dTPT3 for expanding the genetic alphabet in bacteriophages. Based on these results, we next develop and comprehensively optimize an upgraded method for enzymatic preparation of unnatural nucleobase (UB)-containing DNA oligonucleotides with good simplicity and universality.
非天然碱基对（UBP）已被开发用于扩展遗传字母表。UBP dNaM-dTPT3及其类似物已成功用于构建第一套半合成生物体，这表明UBP在生产新型合成生物学部件方面具有巨大潜力。要做到这一点有两个先决条件，一是使用包括DNA聚合酶和连接酶在内的常见工具酶对含有UBP的DNA进行简便操作，二是含有UBP的DNA链易于获得。此外，对于UBP在噬菌体合成生物学中的应用，噬菌体酶对UBP的识别至关重要。在此，我们首先探索噬菌体的B族DNA聚合酶T4 DNA聚合酶D219A对dNaM-dTPT3的识别。引物延伸、稳态动力学和缺口填充实验结果表明，T4 DNA聚合酶D219A能够高效且忠实地复制dNaM-dTPT3，并通过在与dNaM相对的位置插入dTPT3TP或其类似物来高效填充缺口。然后，我们系统地探索了来自噬菌体的不同DNA连接酶对dNaM-dTPT3及其类似物的识别，发现这些DNA连接酶通常能够高效连接dNaM-dTPT3或其类似物旁边的DNA切口，尽管效率略有不同。这些结果表明有更多用于操作dNaM-dTPT3的酶工具，并表明dNaM-dTPT3在噬菌体中扩展遗传字母表的潜在用途。基于这些结果，我们接下来开发并全面优化了一种升级方法，用于酶法制备具有良好简便性和通用性的含非天然核碱基（UB）的DNA寡核苷酸。
