Scientists at The Scripps Research Institute (TSRI) have engineered a bacterium whose genetic material includes an added pair of DNA “letters” (bases) not found in nature.

The research was intended to created new proteins — and even new organisms — that have never existed before.

“Life on Earth in all its diversity is encoded by only two pairs of DNA bases, A-T and C-G, and what we’ve made is an organism that stably contains those two plus a third, unnatural pair of bases,” said TSRI Associate Professor Floyd E. Romesberg, who led the research team.

“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 — from new medicines to new kinds of nanotechnology.”

The report on the achievement appears May 7, 2014, in an advance online publication of the journal Nature.

How to engineer synthetic life

The 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 the natural T-A and C-G base pairs along with the new unnatural base pair that Romesberg’s laboratory had discovered: two molecules known as d5SICS and dNaM.

The goal: get the E. coli cells to replicate this semi-synthetic DNA as normally as possible. To do that, the researchers had to first 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. For that, they used a species of microalgae, that could import the unnatural triphosphates.

The team found, somewhat to their surprise, that the semi-synthetic 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.

The next step will be to demonstrate the in-cell transcription of the new, expanded-alphabet DNA into the RNA that feeds the protein-making machinery of cells. “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,” Romesberg said. “Other applications, such as nanomaterials, are also possible.”

What about risks of a runaway new life form? According to Denis A. Malyshev, a member of the Romesberg laboratory who was lead author of the new report, “the new bases can only get into the cell if we turn on the ‘base transporter’ protein. Without this transporter or when new bases are not provided, the cell will revert back to A, T, G, C, and the d5SICS and dNaM will disappear from the genome.”

Other contributors to the paper, “A semi-synthetic organism with an expanded genetic alphabet,” were Kirandeep Dhami, Thomas Lavergne and Tingjian Chen of TSRI, and Nan Dai, Jeremy M. Foster and Ivan R. Corrêa Jr. of New England Biolabs, Inc.

The research was funded in part by the National Institutes of Health.

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Abstract of Nature paper

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). We 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-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. 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. Here we 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. Lastly, we 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.