Chemical biology: DNA's new alphabet

When Steven Benner set out to re-engineer genetic molecules, he didn't think much of DNA. The first thing you realize is that it is a stupid design, says Benner, a biological chemist at the Foundation for Applied Molecular Evolution in Gainesville, Florida.

Take DNA's backbone, which contains repeating, negatively charged phosphate groups. Because negative charges repel each other, this feature should make it harder for two DNA strands to stick together in a double helix. Then there are the two types of base-pairing: adenine (A) to thymine (T) and cytosine (C) to guanine (G). Both pairs are held together by hydrogen bonds, but those bonds are weak and easily broken up by water, something that the cell is full of. You're trusting your valuable genetic inheritance that you're sending on to your children to hydrogen bonds in water? says Benner. If you were a chemist setting out to design this thing, you wouldn't do it this way at all.

Life may have had good reasons for settling on this structure, but that hasn't stopped Benner and others from trying to change it. Over the past few decades, they have tinkered with DNA's basic building blocks and developed a menagerie of exotic letters beyond A, T, C and G that can partner up and be copied in similar ways. But the work has presented one goddamn problem after another, says Benner. So far, only a few of these unnatural base pairs can be inserted into DNA consecutively, and cells are still not able to fully adopt the foreign biochemistry.

The re-engineering of DNA, and its cousin RNA, has practical goals. Artificial base pairs are already used to detect viruses and may find other uses in medicine. But scientists are also driven by the sheer novelty of it all. Eventually, they hope to develop organisms with an expanded genetic alphabet that can store more information, or perhaps ones driven by a genome with no natural letters at all. In creating these life forms, researchers could learn more about the fundamental constraints on the structure of genetic molecules and determine whether the natural bases are necessary for life or simply one solution of many. Earth has done it a certain way in its biology, says Gerald Joyce, a nucleic-acid biochemist at the Scripps Research Institute in La Jolla, California. But in principle there are other ways to achieve those ends.

Benner first became interested in those other ways as a graduate student in the 1970s. Chemists had synthesized everything from peptides to poisons, and some were trying to build molecules that could accomplish the same functions as natural enzymes or antibodies with different chemical structures. But DNA was largely ignored, he recalls. Chemists were looking at every other class of molecule from a design perspective except the one at the centre of biology, says Benner.

In 1986, Benner started a lab at the Swiss Federal Institute of Technology in Zurich and began to rebuild DNA's backbone. He quickly realized that what seemed like a flaw might be a feature. When he and his team replaced the backbone's negatively charged phosphates with neutral chemical groups1, they found that any strand longer than about a dozen units folded up on itself probably because repelling charges were needed to keep the molecule stretched out.

The bases proved more amenable to tinkering. Benner set out to create base pairs that are similar to nature's, but with rearranged hydrogen bonding units.

His team tested two new pairs: iso-C and iso-G (ref. 2) and and xanthosine3. It showed that polymerase enzymes which copy DNA or transcribe it into RNA could read DNA containing the unnatural bases and insert the complementary partners into a growing DNA or RNA strand. Ribosomes, the cellular machines that 'translate' RNA into protein, could also read an RNA snippet containing iso-C and use it to add an unnatural amino acid to a growing protein4. The base pairing, which is at the centre of genetics, turned out to be for us the most malleable part of the molecule, says Benner. The researchers did encounter a problem, however. Because its hydrogen atoms tend to move around, iso-G often morphed into a different form and paired with T instead of iso-C.

Eric Kool, a chemist now at Stanford University in California, wondered whether his team could develop unnatural bases with fixed hydrogen-bonding arrangements. He and his colleagues made a base similar to the natural base T, but with fluorine in place of the oxygen atoms (see 'Designer DNA'), among other differences5. The structure of the new base, called difluorotoluene (designated F), mimicked T's shape almost exactly but discouraged hydrogen from jumping.

The team soon discovered that F was actually terrible at hydrogen bonding5, but polymerases still treated it like a T: during DNA copying, they faithfully inserted A opposite F (ref. 6) and vice versa7. The work suggested that as long as the base had the right shape, a polymerase could slot it in correctly. If the key fits, it works, says Kool.

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Chemical biology: DNA's new alphabet