Expanding the Genetic Alphabet

Scientists inserted an unnatural base pair, marked X-Y, into the sequence of a plasmid.Image by the researchers, courtesy of Nature

Scientists have created the first living organism that can grow and reproduce using DNA base pairs that aren’t found in nature. The achievement is a major step toward creating novel therapeutics and nanomaterials.

Synthetic biology aims to redesign natural biological systems for new purposes. By reappropriating naturally occurring stop codons, researchers have previously been able to incorporate synthetic non-standard amino acids, the building blocks of proteins, into protein structures in living cells. That accomplishment raises the possibility that scientists might be able to retool nature to create new forms of proteins for therapeutic and other uses.

A team led by Dr. Floyd E. Romesberg of the Scripps Research Institute aimed to expand the genetic code itself, thereby enabling greater flexibility toward creating new biomaterials. DNA is a long molecule made by stringing 4 chemical bases together: adenine (A), guanine (G), cytosine (C), and thymine (T). Certain bases pair with each other—A with T and C with G—to form units called base pairs. The order of these bases defines genes and other information that the cell needs to function.

In the laboratory, researchers have been able to expand the genetic alphabet to include several unnatural base pairs in DNA. But incorporating unnatural bases into DNA within an organism poses bigger challenges. First, bases are brought into the cell by proteins called nucleotide triphosphate transporters, and these proteins would need to be able to carry in the unnatural bases. Second, the unnatural base pair would need to be formed during DNA replication like a natural one and line up stably alongside natural pairs in DNA. The unnatural pair would need to bind with an affinity similar to that of natural pairs in order to separate and rejoin during DNA operations. The bases would also have to avoid being recognized and removed by natural DNA repair mechanisms.

Romesberg’s team chose the unnatural base pair formed between the molecules d5SICS and dNaM for this study. They first tested several different nucleotide triphosphate transporters to find one that could efficiently import the unnatural bases into Escherichia coli cells. Their work was funded by NIH’s National Institute of General Medical Sciences (NIGMS). The study was published on May 15, 2014, in Nature.

The team identified a transporter from a species of microalgae that could import the unnatural bases from the surrounding environment into the cell. They then created a strain of E. coli that made the transporter. Finally, they inserted a plasmid—a circular piece of DNA—that contained one d5SICS-dNaM pair into the cells.

The resulting bacterium is the first organism able to stably maintain DNA comprised of 3 types of base pairs. The plasmid containing the d5SICS–dNaM pair was replicated with a fidelity (retention per doubling) of more than 99%, comparable to the error rate of DNA replication with some natural polymerases. The unnatural base pair wasn’t removed by DNA repair pathways, and the bacteria appeared to grow at rates comparable to those without the synthetic system.

“Life on Earth in all its diversity is encoded by only 2 pairs of DNA bases, A-T and C-G, and what we’ve made is an organism that stably contains those 2 plus a third, unnatural pair of bases,” Romesberg says. “In principle, we could encode new proteins made from new, unnatural amino acids—which would give us greater power than ever to tailor protein therapeutics and diagnostics and laboratory reagents to have desired functions.”

—by Harrison Wein, Ph.D.