A Semi-Synthetic Organism with an Expanded Genetic Alphabet

Organisms are defined by the information encoded in their genomes, and since the evolution of life, this information has been encoded using a two base pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs)(1–3). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM, Fig. 1a), which is efficiently PCR amplified(1) and transcribed(4,5) in vitro, and whose unique mechanism of replication has been characterized(6,7). However, expansion of a organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must enter the cell; endogenous polymerases must be able to faithfully incorporate the unnatural triphosphates into DNA within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into E. coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a significant growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to stably propagate an expanded genetic alphabet.