Life created with two synthetic bases for 6 DNA letters with Original Four G, T, C, A and now X and Y and this expands amino acids from 20 to 172

From the moment life gained a foothold on Earth the diversity of organisms has been written in a DNA code of four letters. The latest study moves life beyond G, T, C and A – the molecules or bases that pair up in the DNA helix – and introduces two new letters of life: X and Y.

Romesberg started out with E coli, a bug normally found in soil and carried by people. Into this he inserted a loop of genetic material that carried normal DNA and two synthetic DNA bases. Though known as X and Y for simplicity, the artificial DNA bases have much longer chemical names, which themselves abbreviate to d5SICS and dNaM.

In living organisms, G, T, C and A come together to form two base pairs, G-C and T-A. The extra synthetic DNA forms a third base pair, X-Y. These base pairs are used to make genes, which cells use as templates for making proteins.

Romesberg found that when the modified bacteria divided they passed on the natural DNA as expected. But they also replicated the synthetic code and passed that on to the next generation. That generation of bugs did the same.

Two new Bases that replicate in living cells, 152 new amino acids, more new proteins

“What we have now, for the first time, is an organism that stably harbours a third base pair, and it is utterly different to the natural ones,” Romesberg said. For now the synthetic DNA does not do anything in the cell. It just sits there. But Romesberg now wants to tweak the organism so that it can put the artificial DNA to good use.

Nature – A semi-synthetic organism with an expanded genetic alphabet

[Wall Street Journal] The experiment demonstrates the feasibility of life-forms based on a different DNA code, independent experts said. Eventually, scientists could use an expanded genetic code to design living cells that could make new medical compounds. By one recent estimate, the market for biologic and protein-based therapies is expected to reach $165 billion a year by 2018.

“Most people thought this wasn’t possible,” said biochemist Steven Benner at the Foundation for Applied Molecular Evolution in Gainesville, Fla., who wasn’t involved in the project. Many scientists assumed that a normal cell would ignore any imitation DNA. “He has gone inside a cell and gotten it to work and that is a shock,” said Dr. Benner.

With growing mastery, scientists have been tinkering with this natural information-storage system that is found inside every cell. They routinely cut and splice normal DNA to alter plants, bacteria and animals. They have used its ultraminiature storage capacity to encode books, poems and popular music. They even have programmed DNA to perform computer-like calculations.

Hoping to make DNA even more capable, scientists working in a new field called synthetic biology have been trying to perfect alternative genetic codes containing up to 12 DNA characters. Until now, researchers haven’t been able to make the artificial DNA work in a living, reproducing organism.

In their work, Dr. Romesberg and his team sidestepped a technical problem that had stymied other scientists. They didn’t try to create cells that could produce unnatural DNA on their own. Instead, they fed a chemical supplement to bacteria—a common laboratory strain of Escherichia coli—to give the cells some help.

Supplied with the dietary supplement, the bacteria readily replicated the artificial DNA, neatly combining the two synthetic characters into a new base pair, and then built that unnatural base pair into the structure of its DNA molecules, the scientists said.

By design, the new DNA didn’t change the basic functions of the altered cells, though they did grow slightly more slowly, the scientists said.

The technique also builds in a safety feature. Without the chemical additive, the bacteria stop making the artificial DNA and revert to normal.

[Science Magazine] – Getting live bacteria to replicate altered DNA was another challenge entirely. The bacteria would need either to synthesize the new genetic letters themselves or to import them from the surrounding culture medium. In algae, Romesberg and his colleagues identified a protein that grabs nucleotide bases and pulls them into the cell. They spliced the gene for this transporter protein into Escherichia coli bacteria and found it enabled the bacteria to pull in presynthesized X and Y bases as well. The team had also engineered their E. coli to harbor small rings of DNA called plasmids carrying X-Y pairs. When the bacteria copied those plasmids, they used the newly imported X and Y bases—yet the engineered cells grew just as well as their native cousins.

Next, Romesberg says he hopes to use his expanded genetic alphabet to create designer proteins. Scripps biochemist Peter Schultz and others have already engineered bacteria to build proteins with dozens of amino acids beyond nature’s standard 20. But those experiments use natural DNA to code for unnatural amino acids. The newly expanded genetic alphabet, Thyer says, should yield a vastly more diverse menu of proteins with a wide variety of new chemical functions, such as medicines better able to survive in the body and protein-based materials that assemble themselves. Romesberg says forays into that new world of proteins are already under way.


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.

3 pages of supplemental information

SOURCES – Nature, Wall Street Journal, Guardian UK, Science Magazine

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