ScienceDaily — 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.
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. (Credit: © Dmitry Sunagatov / Fotolia)
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 replication."
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 DNA alphabet -- 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 DNA 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."
The other contributors to the paper, "KlenTaq polymerase replicates
unnatural base pairs by inducing a Watson-Crick geometry," are Thomas
Lavergne of the Romesberg lab, Wolfram Welte and Kay Diederichs of the
Marx lab, and Phillip Ordoukhanian of the Center for Protein and Nucleic
Acid Research at The Scripps Research Institute.
The study was supported in part by a grant from the National Institutes of Health.
Journal Reference:
- Karin Betz, Denis A Malyshev, Thomas Lavergne, Wolfram Welte, Kay Diederichs, Tammy J Dwyer, Phillip Ordoukhanian, Floyd E Romesberg & Andreas Marx. KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry. Nature Chemical Biology, 03 June 2012 DOI: 10.1038/nchembio.966
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