Study suggests expanding the genetic alphabet may be easier than previously thought

June 3, 2012

A new study led by scientists at The Scripps Research Institute suggests that the replication process for DNA—the genetic instructions for living organisms that is composed of four bases (C, G, A and T)—is more open to unnatural letters than had previously been thought. An expanded "DNA alphabet" could carry more information than natural DNA, potentially coding for a much wider range of molecules and enabling a variety of powerful applications, from precise molecular probes and nanomachines to useful new life forms.

The new study, which appears in the June 3, 2012 issue of Nature Chemical Biology, solves the mystery of how a previously identified pair of artificial DNA bases can go through the DNA replication process almost as efficiently as the four natural bases.

"We now know that the efficient replication of our unnatural base pair isn't a fluke, and also that the replication process is more flexible than had been assumed," said Floyd E. Romesberg, associate professor at Scripps Research, principal developer of the new DNA bases, and a senior author of the new study. The Romesberg laboratory collaborated on the new study with the laboratory of co-senior author Andreas Marx at the University of Konstanz in Germany, and the laboratory of Tammy J. Dwyer at the University of San Diego.

Adding to the DNA Alphabet

Romesberg and his lab have been trying to find a way to extend the DNA alphabet since the late 1990s. In 2008, they developed the efficiently replicating bases NaM and 5SICS, which come together as a complementary base pair within the DNA helix, much as, in normal DNA, the base adenine (A) pairs with thymine (T), and cytosine (C) pairs with guanine (G).

The following year, Romesberg and colleagues showed that NaM and 5SICS could be efficiently transcribed into RNA in the lab dish. But these bases' success in mimicking the functionality of natural bases was a bit mysterious. They had been found simply by screening thousands of synthetic nucleotide-like molecules for the ones that were replicated most efficiently. And it had been clear immediately that their chemical structures lack the ability to form the hydrogen bonds that join natural base pairs in DNA. Such bonds had been thought to be an absolute requirement for successful DNA replication —a process in which a large enzyme, DNA polymerase, moves along a single, unwrapped DNA strand and stitches together the opposing strand, one complementary base at a time.

An early structural study of a very similar base pair in double-helix DNA added to Romesberg's concerns. The data strongly suggested that NaM and 5SICS do not even approximate the edge-to-edge geometry of natural base pairs—termed the Watson-Crick geometry, after the co-discoverers of the DNA double-helix. Instead, they join in a looser, overlapping, "intercalated" fashion. "Their pairing resembles a 'mispair,' such as two identical bases together, which normally wouldn't be recognized as a valid base pair by the DNA polymerase," said Denis Malyshev, a graduate student in Romesberg's lab who was lead author along with Karin Betz of Marx's lab.

Yet in test after test, the NaM-5SICS pair was efficiently replicable. "We wondered whether we were somehow tricking the DNA polymerase into recognizing it," said Romesberg. "I didn't want to pursue the development of applications until we had a clearer picture of what was going on during replication."

Edge to Edge

To get that clearer picture, Romesberg and his lab turned to Dwyer's and Marx's laboratories, which have expertise in finding the atomic structures of DNA in complex with DNA polymerase. Their structural data showed plainly that the NaM-5SICS pair maintain an abnormal, intercalated structure within double-helix DNA—but remarkably adopt the normal, edge-to-edge, "Watson-Crick" positioning when gripped by the polymerase during the crucial moments of DNA replication.

"The DNA polymerase apparently induces this unnatural base pair to form a structure that's virtually indistinguishable from that of a natural base pair," said Malyshev.

NaM and 5SICS, lacking hydrogen bonds, are held together in the DNA double-helix by "hydrophobic" forces, which cause certain molecular structures (like those found in oil) to be repelled by water molecules, and thus to cling together in a watery medium. "It's very possible that these hydrophobic forces have characteristics that enable the flexibility and thus the replicability of the NaM-5SICS base pair," said Romesberg. "Certainly if their aberrant structure in the double helix were held together by more rigid covalent bonds, they wouldn't have been able to pop into the correct structure during DNA ."

An Arbitrary Choice?

The finding suggests that NaM-5SICS and potentially other, hydrophobically bound base pairs could some day be used to extend the DNA alphabet. It also hints that Evolution's choice of the existing four-letter —on this planet—may have been somewhat arbitrary. "It seems that life could have been based on many other genetic systems," said Romesberg.

He and his laboratory colleagues are now trying to optimize the basic functionality of NaM and 5SICS, and to show that these new bases can work alongside natural bases in the of a living cell.

"If we can get this new base pair to replicate with high efficiency and fidelity in vivo, we'll have a semi-synthetic organism," Romesberg said. "The things that one could do with that are pretty mind blowing."

Explore further: DNA size a crucial factor in genetic mutations, study finds

More information: "KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry," Nature Chemical Biology, 2012.

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2.3 / 5 (3) Jun 03, 2012
"The things that one could do with that are pretty mind blowing." Like creating an alien life forms suitable to other planetary conditions?
not rated yet Jun 03, 2012
I have a question about this:
Does the RNA polymerase molecule which stitches together the complementary DNA strand,simply select the matching nucleotide from the surrounding cytoplasm and feed it into a slot [so to speak], or does it actually make up the partner nucleotide on the spot?

If the former is the case then that will be a major limiting factor until the researchers can create genes which code for the manufacture of the novel nucleotides. If the latter is the case then Hello Brave New World!
not rated yet Jun 03, 2012
Like with most molecules needed in biology, they are floating around in some solution. Nucleotides are floating around in nucleoplasm and DNA poly "feeds them in" when replicating.
5 / 5 (3) Jun 03, 2012
It is probably possible to add protein complexes that form a synthesizer the new nucleotides. But if they are hydrophobic, I wonder how soluble they will be in the cytoplasm. They will undoubtedly have to be paired with a hydophilic substance to dissolve, which leaves open questions of how the pair would dissociate before binding to the t-RNA that builds new DNA or RNA intermediates. It looks to me like a lot of work is in front of the researchers, but what they have accomplished so far is utterly amazing. Crudo's for some excellent work!
3.4 / 5 (5) Jun 03, 2012
It also hints that Evolution's choice of the existing four-letter DNA alphabeton this planetmay have been somewhat arbitrary.

purely naturalistic argument;

1, evolution could make no choice.
2, natural selection must have weeded out the other candidate molecules, or else they were deemed inappropriate from the start.
not rated yet Jun 03, 2012

They said the interaction between the interclated areas are hydrophobic, i.e VDW forces. It is extremely unlikely the whole molecule is non-polar. If that were the case, the nucleotides would just associate in a polar solution. I would suspect, without knowing the structural formula of the nucleotide, that it is a polar molecule. Furthermore, since these nucleotides are inserted into DNA they still have to have the standard 5'/3' bonding areas, which again makes me suspect these are polar molecules.
not rated yet Jun 03, 2012
Crudo's for some excellent work!

Are Crudo's more like Kudos or like Croutons? Because I enjoy receiving both.
1 / 5 (1) Jun 05, 2012
Interesting first step, but the real breakthrough will be in using these new bases to build artificial genes that actually work in conjunction with all the other genes on the strand. Replicating DNA that includes these bases is not the same as having the new bases code for novel characteristics in a synthetic organism. Until that is accomplished, we are still talking science fiction here.
not rated yet Jun 30, 2012
This is a very important discovery. Not only for what we could possibly achieve with it, like genes with new compositions and, mainly, new fuctionality.

But also because of the new aspects that we may come to understand on the origins of life, here and also on other planets.

Perhaps, it might be possible to have a DNA, or the equivalent of DNA, with more than 4 pairs of bases. Perhaps we could encode all kinds of information inside such a molecule. In the future, instead of a letter with invisible ink, they could send a cat with a tiny tumor containing the encrypted message.

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