NMPA Key Laboratory for Research and Evaluation of Innovative Drug, China Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, Henan Normal University, Xinxiang, Henan 453007, China.
Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals, School of Chemistry and Chemical Engineering, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, Henan Normal University, Xinxiang, Henan 453007, China.
Bioorg Chem. 2023 Nov;140:106827. doi: 10.1016/j.bioorg.2023.106827. Epub 2023 Aug 30.
The high fidelity poses a central role in developing unnatural base pairs (UBPs), which means the high pairing capacity of unnatural bases with their partners, and low mispairing with all the natural bases. Different strategies have been used to develop higher-fidelity UBPs, including optimizing hydrophobic interaction forces between UBPs. Variant substituent groups are allowed to fine tune the hydrophobic forces of different UBPs' candidates. However, the modifications on the skeleton of TPT3 base are rare and the replication fidelity of TPT3-NaM remains hardly to improve so far. In this paper, we reasoned that modifying and/or expanding the aromatic surface by Bromo-substituents to slightly increase hydrophobicity of TPT3 might offer a way to increase the fidelity of this pair. Based on the hypothesis, we synthesized the bromine substituted TPT3, 2-bromo-TPT3 and 2, 4-dibromo-TPT3 as the new TPT3 analogs. While the enzyme reaction kinetic experiments showed that d2-bromo-TPT3-dNaM pair and d2, 4-dibromo-TPT3TP-dNaM pair had slightly less efficient incorporation and extension rates than that of dTPT3-dNaM pair, the assays did reveal that the mispairing of 2-bromo-TPT3 and 2, 4-dibromo-TPT3 with all the natural bases could dramatically decrease in contrast to TPT3. Their lower mispairing capacity promoted us to run polymerase chain amplification reactions, and a higher fidelity of d2-bromo-TPT3-dNaM pair could be obtained with 99.72 ± 0.01% of the in vitro replication fidelity than that of dTPT3-dNaM pair, 99.52 ± 0.09%. In addition, d2-bromo-TPT3-dNaM can also be effectively copied in E. coli cells, which showed the same replication fidelity as that of dTPT3-dNaM in the specific sequence, but a higher fidelity in the random sequence context.
高保真度在开发非天然碱基对（UBP）中起着核心作用，这意味着非天然碱基与它们的配对碱基具有很高的配对能力，并且与所有天然碱基的错配率很低。已经使用了不同的策略来开发更高保真度的 UBP，包括优化 UBP 之间的疏水相互作用力。允许变体取代基来微调不同 UBP 候选物的疏水相互作用力。然而，迄今为止，对 TPT3 碱基骨架的修饰很少，并且 TPT3-NaM 的复制保真度几乎难以提高。在本文中，我们推断，通过溴取代基略微增加 TPT3 的疏水性，修饰和/或扩展芳基面可能是提高该碱基对保真度的一种方法。基于这一假设，我们合成了溴取代的 TPT3、2-溴-TPT3 和 2,4-二溴-TPT3 作为新的 TPT3 类似物。虽然酶反应动力学实验表明，d2-溴-TPT3-dNaM 对和 d2,4-二溴-TPT3TP-dNaM 对的掺入和延伸速率比 dTPT3-dNaM 对略低，但实验结果表明，2-溴-TPT3 和 2,4-二溴-TPT3 与所有天然碱基的错配率显著降低，与 TPT3 相比。它们较低的错配能力促使我们进行聚合酶链扩增反应，并且 d2-溴-TPT3-dNaM 对的体外复制保真度比 dTPT3-dNaM 对高 99.72 ± 0.01%，达到 99.52 ± 0.09%。此外，d2-溴-TPT3-dNaM 也可以有效地在大肠杆菌细胞中复制，其在特定序列中的复制保真度与 dTPT3-dNaM 相同，但在随机序列背景下的保真度更高。
