Access to Photostability-Enhanced Unnatural Base Pairs via Local Structural Modifications

• Published in: ACS synthetic biology 2022-01, Vol.11 (1), p.334-342
• Main Authors: Wang, Honglei, Wang, Luying, Ma, Nana, Zhu, Wuyuan, Huo, Bianbian, Zhu, Anlian, Li, Lingjun
• Format: Article
• Language: English
• Subjects: Base Pairing - genetics; Escherichia coli - genetics; Escherichia coli - metabolism; Kinetics; Molecular Structure; Polymerase Chain Reaction
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.1c00451

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 E. coli 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.