The Scientist
All life on Earth uses a genetic code based on four nucleotides. Now, scientists have created one with eight.

By combining four synthetic nucleotides with the four found naturally in nucleic acids, researchers have created eight-letter deoxyribonucleic acid molecules that look and behave like the real thing, and are even able to be transcribed into ribonucleic acids, according to a paper in Science today February 21. With twice the information storage capacity of natural nucleic acids, these eight-letter, or “hachimoji” molecules - could have countless biotechnological applications, say scientists.

“This is really an exciting paper . . . a true engineering feat. It elegantly increases the number of deoxyribonucleic acid and ribonucleic acid building blocks and dramatically expands the information density of nucleic acids,” Northwestern University’s Michael Jewett, who was not involved with the research, writes in an email to The Scientist.

“It’s really exciting to see somebody engineer [such] a system,” says biologist Eugene Wu of the University of Richmond who also did not participate in the project. “It begs the question, at the origins of life, why these four [nucleotides formed nucleic acids]? Why couldn’t it have been eight or some other number?”

Whatever the reason, for the last 4 billion years or so, just two base pairs - formed between guanine - G and cytosine - C, and between adenine - A and thymine - T, or in the case of ribonucleic acid, uracil - U - have been all that was required for nature to create the endless variety of life found on Earth. But in theory there could have been more, says project leader Steven Benner of the Foundation for Applied Molecular Evolution and Firebird Biomolecular Sciences in Florida.

A base pair forms when a purine - G or A - connects via hydrogen bonds to a pyrimidine - C, T, or U. Yet there are other purine- and pyrimidine-type structures that could hypothetically connect to produce the same helical structure as standard deoxyribonucleic acid.

Benner has calculated that a total of four additional hydrogen-bonded base pairs, formed from eight novel structures, are possible. Essentially, deoxyribonucleic acid has not exploited its structural limits completely, he says. “And for that reason, the deoxyribonucleic acid molecule could be expanded. . . . You could actually add more letters”.

Benner’s team has previously incorporated two synthetic nucleotides - one base pair, Z and P - into deoxyribonucleic acid and shown that these can be replicated and transcribed in vitro. Now, his team has added another pair, S and B.

The team incorporated the chemically synthesized novel nucleotides into double-stranded oligonucleotides - also containing G, A, T, C, Z, and P - and then tested the molecules’ melting temperatures - the point at which the hydrogen bonds are disrupted to form single-stranded molecules. The observed melting temperatures were on average within 2.1 Celsius of predictions - a similar margin of error for standard deoxyribonucleic acid oligonucleotides.

“What Steve’s now shown is that you can literally double the units that can be incorporated into deoxyribonucleic acid and maintain that [predictable chemistry], which I think is spectacular and . . . a landmark achievement,” says Floyd Romesberg of The Scripps Research Institute in California who was not part of the research team.

Additionally, the high-resolution crystal structures of three different hajimoji deoxyribonucleic acid oligonucleotides confirmed the structural similarity.

Chemically speaking, then, hachimoji deoxyribonucleic acid looks and behaves like standard deoxyribonucleic acid. Enzymes that read and process nucleic acids, however, are hard to fool, so to transcribe hachimoji deoxyribonucleic acid into ribonucleic acid - a test of its information-transmitting ability - the team tried a number of bacteriophage ribonucleic acid polymerase variants until they found one capable of the task.

Using this ribonucleic acid polymerase the team transcribed a hachimoji version of a known ribonucleic acid aptamer - called spinach - that binds and illuminates a particular fluorophore. Sure enough, the transcribed hachimoji ribonucleic acid glowed as expected.

The ability to make functional hachimoji ribonucleic acids “opens up a lot of possibilities in the ribonucleic acid biotechnology field,” says biological chemist Nigel Richards of Cardiff University who did not participate in the research. ribonucleic acids can be catalytic molecules, he explains, so with a new palette of nucleotides, “you have more functional groups that can make different types of interactions with their target molecules . . . [increasing] the range of catalysis that you can do.”

Hachimoji deoxyribonucleic acid could even be combined with other types of artificial nucleotides that are based on a different base-pairing chemistry, thus potentially increasing functionality yet further, says Ichiro Hirao of the Institute of Biotechnology and Nanotechnology in Singapore who was not involved with the research.

There really are, says Romesberg, “an uncountable number of applications.”