Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet

We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding but by hydrophobic and packing forces. These UBPs enabled the creation of the first semisynthetic organisms (SSOs) that store increased genetic information and use it to produce proteins containing noncanonical amino acids. However, retention of the UBPs was poor in some sequence contexts. Here, to optimize the SSO, we synthesize two novel benzothiophene-based dNaM analogs, dPTMO and dMTMO, and characterize the corresponding UBPs, dPTMO-dTPT3 and dMTMO-dTPT3. We demonstrate that these UBPs perform similarly to, or slightly worse than, dNaM-dTPT3 in vitro. However, in the in vivo environment of an SSO, retention of dMTMO-dTPT3, and especially dPTMO-dTPT3, is significantly higher than that of dNaM-dTPT3. This more optimal in vivo retention results from better replication, as opposed to more efficient import of the requisite unnatural nucleoside triphosphates. Modeling studies suggest that the more optimal replication results from specific internucleobase interactions mediated by the thiophene sulfur atoms. Finally, we show that dMTMO and dPTMO efficiently template the transcription of RNA containing TPT3 and that their improved retention in DNA results in more efficient production of proteins with noncanonical amino acids. This is the first instance of using performance within the SSO as part of the UBP evaluation and optimization process. From a general perspective, the results demonstrate the importance of evaluating synthetic biology parts in their in vivo context and further demonstrate the ability of hydrophobic and packing interactions to replace the complementary hydrogen bonding that underlies the replication of natural base pairs. From a more practical perspective, the identification of dMTMO-dTPT3 and especially dPTMO-dTPT3 represents significant progress toward the development of SSOs with an unrestricted ability to store and retrieve increased information.