When chemical biologists began adding new, unnatural base pairs to DNA’s A-T and G-C genetic alphabet, they risked objections of the sort expressed by Shakespeare:

To gild refined gold, to paint the lily, to throw a perfume on the violet, to smooth the ice, or add another hue unto the rainbow, or with taper-light to seek the beauteous eye of heaven to garnish, is wasteful and ridiculous excess.

The chemical biologists persisted, however, arguing that only by pushing DNA to its structural limits could nature’s design be properly appreciated. In addition, some scientists argued, artificial genetic systems could enrich evolutionary biology, synthetic biology, and astrobiology. Finally, those possessing the audacity to tinker with nature’s underlying code pointed to practical benefits—applications from new medicines to new kinds of nanotechnology.

Whatever the justification, chemical biologists can now boast another advance: Unnatural base pairs, once curiosities confined to in vitro systems, have now been integrated into a life form, the common bacterium E. coli. The cells of the re-engineered bacterium can replicate their unnatural DNA bases more or less normally, for as long as the molecular building blocks are supplied.

This feat was revealed May 7 in Nature, in an article entitled “A semi-synthetic organism with an expanded genetic alphabet.” The article’s authors, led by Floyd E. Romesberg, Ph.D., an associate professor at The Scripps Research Institute (TSRI), extended work that had previously introduced unnatural base pairs that could hook up across a double-strand of DNA almost as snugly as natural base pairs. This earlier work had also showed that DNA containing these unnatural base pairs could replicate in the presence of the right enzymes. Further research had even shown that the semi-synthetic DNA could be transcribed into RNA.

Expansion of a living organism’s genetic alphabet, however, was another matter. As indicated in the current Nature article, such an advance would pose a litany of 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 unnatural base pairs within the complex cellular milieu; and finally, the unnatural base pairs must be stable in the presence of pathways that maintain the integrity of DNA.”

Undaunted, the TSRI team synthesized a stretch of circular DNA known as a plasmid and inserted it into cells of the common bacterium E. coli. The plasmid DNA contained natural T-A and C-G base pairs along with the best-performing unnatural base pair Romesberg's laboratory had discovered, two molecules known as d5SICS and dNaM. The goal was to get the E. coli cells to replicate this semisynthetic DNA as normally as possible.

The greatest hurdle may be reassuring to those who fear the uncontrolled release of a new life form: the molecular building blocks for d5SICS and dNaM are not naturally in cells. Thus, to get the E. coli to replicate the DNA containing these unnatural bases, the researchers had to supply the molecular building blocks artificially, by adding them to the fluid solution outside the cell. Then, to get the building blocks, known as nucleoside triphosphates, into the cells, they had to find special triphosphate transporter molecules that would do the job.

The researchers eventually were able to find a triphosphate transporter, made by a species of microalgae, that was good enough at importing the unnatural triphosphates. The TSRI team found, somewhat to their surprise, that the semisynthetic plasmid replicated with reasonable speed and accuracy, did not greatly hamper the growth of the E. coli cells, and showed no sign of losing its unnatural base pairs to DNA repair mechanisms.

Reflecting on this achievement, Dr. Romesberg said, “Life on Earth in all its diversity is encoded by only two pairs of DNA bases, and what we’ve made is an organism that stably contains those two plus a third unnatural pair of bases. This shows that other solutions to storing information are possible and, of course, takes us closer to an expanded-DNA biology that will have many exciting applications.”

Such applications were considered in the Nature article: “In the future, this organism, or a variant with the unnatural base pairs incorporated at other episomal or chromosomal loci, should provide a synthetic biology platform to orthogonally re-engineer cells, with applications ranging from site-specific labeling of nucleic acids in living cells to the construction of orthogonal transcription networks and eventually the production and evolution of proteins with multiple, different unnatural amino acids.”

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