Life created A, C, G, and T. Now, say hello to X and Y.
Organisms are defined by the information encoded in their genomes, and since the origin 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, 2, 3. Scientists have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS–dNaM), which is efficiently PCR-amplified1 and transcribed4, 5 in vitro, and whose unique mechanism of replication has been characterized6, 7. However, expansion of an organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA containing the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA.
In a new study, Scripps scientists show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia 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 notable growth burden. They find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
The history of these new letters—which the scientists call X and Y—can be traced back to 1998 when Rosmeberg and his colleagues first tinkered with the idea. They sought to develop a pair of genetic letters that were similar enough to the letters life already uses that they could function (and wouldn’t be rejected by) the existing framework of DNA. Yet the letters had to be different enough that they wouldn’t be accidentally mispaired with the original four letters—and effectively forgotten.
The letters saw many, many failed incarnations before they morphed into what the researchers presented today. “After 14 years we had developed, made, and optimized over 300 of these nucleotides,” Rosmeberg says. Then came the bigger hurdle: getting a real cell to reproduce X and Y after scientists spliced them in. While cells come stock with the machinery to churn out the original four letters, the scientists had no way to easily alter their bacteria to likewise synthesize X and Y.
“This is a very major accomplishment in our efforts to inch towards a synthetic biology," says Steven Benner, a synthetic biologist at the Foundation for Applied Molecular Evolution who was not involved in the study. "Many in the broader community thought that Floyd's result would be impossible to achieve."