Le Anthony V, Hartman Matthew C T
Virginia Commonwealth University, Department of Chemistry 1001 W Main St. Richmond VA 23284 USA
Virginia Commonwealth University, Massey Cancer Center 401 College St. Richmond VA 23219 USA.
RSC Chem Biol. 2024 Sep 11;5(11):1111-21. doi: 10.1039/d4cb00121d.
Unnatural base pairs (UBP) promise to diversify cellular function through expansion of the genetic code. Some of the most successful UBPs are the hydrophobic base pairs 5SICS:NaM and TPT3:NaM developed by Romesberg. Much of the research on these UBPs has emphasized strategies to enable their efficient replication, transcription and translation in living organisms. These experiments have achieved spectacular success in certain cases; however, the complexity of working places strong constraints on the types of experiments that can be done to optimize and improve the system. Testing UBPs , on the other hand, offers advantages including minimization of scale, the ability to precisely control the concentration of reagents, and simpler purification of products. Here we investigate the orthogonality of NaM-containing base pairs in transcription and translation, looking at background readthrough of NaM codons by the native machinery. We also describe an improved synthesis of NaM triphosphate (NaM-TP) and a new assay for testing the purity of UBP containing RNAs.
非天然碱基对（UBP）有望通过扩展遗传密码来使细胞功能多样化。一些最成功的非天然碱基对是由罗姆斯伯格开发的疏水碱基对5SICS:NaM和TPT3:NaM。对这些非天然碱基对的许多研究都强调了使其在活生物体中有效复制、转录和翻译的策略。在某些情况下，这些实验取得了惊人的成功；然而，实际操作的复杂性对为优化和改进该系统而可进行的实验类型施加了严格限制。另一方面，测试非天然碱基对具有诸多优势，包括规模最小化、能够精确控制试剂浓度以及产物纯化更简便。在此，我们研究含NaM碱基对在转录和翻译中的正交性，观察天然机制对NaM密码子的背景通读情况。我们还描述了三磷酸NaM（NaM-TP）的改进合成方法以及一种用于测试含非天然碱基对RNA纯度的新检测方法。
