Wang Honglei, Wang Luying, Ma Nana, Zhu Wuyuan, Huo Bianbian, Zhu Anlian, Li Lingjun
Henan Key Laboratory of Organic Functional Molecule and Drug Innovation, 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.
ACS Synth Biol. 2022 Jan 21;11(1):334-342. doi: 10.1021/acssynbio.1c00451. Epub 2021 Dec 10.
Completing the storage and retrieval of increased genetic information in vivo and producing therapeutic proteins have been achieved by the unnatural base pair dNaM-dTPT3. Up to now, some biological and chemical approaches are implemented to improve the semi-synthetic organism (SSO). However, the photosensitivity of this pair, suggested as a potential threat to the healthy growth of cells, is still a problem to solve. Hence, we designed and synthesized a panel of TPT3 analogues with the basic structural skeletons of TPT3 but modified thiophene rings at variant sites to improve the photostability of unnatural base pairs. A comprehensive screening strategy, including photosensitivity tests, kinetic experiments, and replication in vitro by PCR and in vivo by amplification, was implemented. A new pair, dNaM-dTAT1, which had almost equally high efficiency and fidelity with the dNaM-dTPT3 pair itself both in vivo and in vitro, was proven to be more photostable and thermostable and less toxic to cells. The discovery of dNaM-dTAT1 represents our first progress for the optimization of this type of bases toward more photostable properties; our data also suggest that less photosensitive unnatural base pairs will be beneficial to build a healthier cellular replication system.
通过非天然碱基对dNaM-dTPT3已实现了在体内完成增加的遗传信息的存储和检索以及生产治疗性蛋白质。到目前为止，已经实施了一些生物学和化学方法来改进半合成生物体（SSO）。然而，这对碱基的光敏性被认为是对细胞健康生长的潜在威胁，仍然是一个有待解决的问题。因此，我们设计并合成了一组具有TPT3基本结构骨架但在不同位点修饰噻吩环的TPT3类似物，以提高非天然碱基对的光稳定性。实施了一种全面的筛选策略，包括光敏性测试、动力学实验以及通过PCR进行体外复制和通过扩增进行体内复制。一种新的碱基对dNaM-dTAT1，在体内和体外与dNaM-dTPT3碱基对本身具有几乎同样高的效率和保真度，被证明具有更高光稳定性和热稳定性，并且对细胞毒性更小。dNaM-dTAT1的发现代表了我们在优化这类碱基以获得更高光稳定性方面的首个进展；我们的数据还表明，光敏性更低的非天然碱基对将有利于构建更健康的细胞复制系统。
