Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase
Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase
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DOI：
10.1021/acssynbio.2c00456
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发表时间：
2022-12-01
影响因子：
4.7
通讯作者：
Chen，Tingjian
中科院分区：
文献类型：
--
作者：
Wang，Guangyuan;He，Chuanping;Chen，Tingjian
非天然碱基对（UBPs）的发现极大地扩展了体外和体内的遗传字母表，并在生物技术和生物制药工业中产生了许多潜在的应用。高效合成含有非天然碱基的寡核苷酸无疑是充分利用UBP的必要前提，而利用末端脱氧核苷酸转移酶（TdTs）进行寡核苷酸的从头合成已成为克服传统固相合成局限性的一种极具潜力的方法。在此，我们报告了非天然核碱基dTPT 3和dNaM的核苷酸的有效的模板非依赖性掺入，其被设计为使迄今为止最成功的UBP之一dTPT 3-dNaM在优化的条件下用TdT酶掺入DNA寡核苷酸中。我们还证明了具有不同功能接头的dTPT 3衍生物有效的TdT掺入寡核苷酸中用于核酸的正交标记及其应用。还描述了在TdT的协助下，在任意地点用UBP进行DNA的日常实验室制备的方法的发展。
Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet bothin vitroandin vivoand led to numerous potential applications in the biotechnology and biopharmaceutical industry. Efficient synthesis of oligonucleotides containing unnatural nucleobases is undoubtedly an essential prerequisite for making full use of the UBPs, andde novosynthesis of oligonucleotides with terminal deoxynucleotidyl transferases (TdTs) has emerged as a method of great potential to overcome limitations of traditional solid-phase synthesis. Herein, we report the efficient template-independent incorporation of nucleotides of unnatural nucleobases dTPT3 and dNaM, which have been designed to make one of the most successful UBPs to date, dTPT3-dNaM, into DNA oligonucleotides with a TdT enzyme under optimized conditions. We also demonstrate the efficient TdT incorporation of dTPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof. The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.