Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet

We have developed an unnatural base pair (UBP) and a semisynthetic organism (SSO) that imports the constituent unnatural nucleoside triphosphates and uses them to replicate DNA containing the UBP. However, propagation of the UBP is at least in part limited by the stability of the unnatural triphosphates, which are degraded by cellular and secreted phosphatases. To circumvent this problem, we now report the synthesis and evaluation of unnatural triphosphates with their beta, gamma-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTP(CF2) and dTPT3TP(CF2). We find that although dNaMTP(CF2) cannot support in vivo replication, likely due to poor polymerase recognition, dTPT3TP(CF2) can, and moreover, its increased stability can contribute to increased UBP retention. The data demonstrate the promise of this chemical approach to SSO optimization, and suggest that other modifications should be sought that confer phosphatase resistance without interfering with polymerase recognition.