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. 2022 Dec 16;11(12):4142-4155. doi: 10.1021/acssynbio.2c00456. Epub 2022 Dec 1.
Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet both and and 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, and synthesis 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.
非天然碱基对（UBPs）的发展极大地扩展了遗传密码子，并且在生物技术和生物制药行业中带来了众多潜在的应用。含有非天然碱基的寡核苷酸的高效合成无疑是充分利用 UBPs 的必要前提，而末端脱氧核苷酸转移酶（TdT）合成的寡核苷酸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，我们报告了在优化条件下，TdT 酶能够有效地将经过设计以构建迄今为止最成功的 UBP 之一 dTPT3-dNaM 的非天然碱基 dTPT3 和 dNaM 的核苷酸模板独立地掺入到 DNA 寡核苷酸中。我们还展示了具有不同功能连接子的 dTPT3 衍生物在寡核苷酸中的有效 TdT 掺入，用于核酸的正交标记及其应用。此外，还描述了在 TdT 的辅助下，在任意位置以日常实验室方式制备含有 UBP 的 DNA 的方法。
