[tt] [nano] newscientist: artificial DNA base-pair replicated by natural polymerase

[Alejandro Dubrovsky]

(
http://technology.newscientist.com/article/dn13252-artificial-letters-added-to-lifes-alphabet.html
)

Artificial letters added to life's alphabet

 * 13:07 30 January 2008
 * NewScientist.com news service
 * Robert Adler


Two artificial DNA "letters" that are accurately and efficiently
replicated by a natural enzyme have been created by US researchers.
Adding the two artificial building blocks to the four that naturally
comprise DNA could allow wildly different kinds of genetic engineering,
they say.

Eventually, the researchers say, they may be able to add them into the
genetic code of living organisms.

The diversity of life on earth evolved using genetic code made from
arrangements of four genetic "bases", sometimes described as letters.
They are divided into two pairs, which bond together from opposite
strands of a DNA molecule to form the rungs of its characteristic
double-helix shape.

The unnatural but functional new base pair is the fruit of nearly a
decade of research by chemical biologist Floyd Romesberg, at the Scripps
Research Institute, La Jolla, California, US.

Romesberg and colleagues painstakingly created a library of nearly 200
potential new genetic bases that are slight variations on the natural
ones. Unfortunately, none of them were similar enough in structure and
chemistry to the real thing to be copied accurately by the polymerase
enzymes that replicate DNA inside cells.
Random generation

Frustrated by the slow pace designing and synthesising potential new
bases one at a time, Romesberg borrowed some tricks from drug
development companies. The resulting large scale experiments generated
many potential bases at random, which were then screened to see if they
would be treated normally by a polymerase enzyme.

With the help of graduate student Aaron Leconte, the group synthesized
and screened 3600 candidates. Two different screening approaches turned
up the same pair of molecules, called dSICS and dMMO2.

The molecular pair that worked surprised Romesberg. "We got it and said,
'Wow!' It would have been very difficult to have designed that pair
rationally."

But the team still faced a challenge. The dSICS base paired with itself
more readily than with its intended partner, so the group made minor
chemical tweaks until the new compounds behaved properly.
Novel DNA

"We probably made 15 modifications," says Romesberg, "and 14 made it
worse." Sticking a carbon atom attached to three hydrogen atoms onto the
side of dSICS, changing it to d5SICS, finally solved the problem. "We
now have an unnatural base pair that's efficiently replicated and
doesn't need an unnatural polymerase," says Romesberg. "It's staring to
behave like a real base pair."

The team is now eager to find out just what makes it work. "We still
don't have a detailed understanding of how replication happens," says
Romesberg. "Now that we have an unnatural base pair, we are continuing
experiments to understand it better."

In the near future, Romesberg expects the new base pairs will be used to
synthesize DNA with novel and unnatural properties. These might include
highly specific primers for DNA amplification; tags for materials, such
as explosives, that could be detected without risk of contamination from
natural DNA; and building novel DNA-based nanomaterials.
Increased 'evolvability'

More generally, Romesberg notes that DNA and RNA are now being used for
hundreds of purposes: for example, to build complex shapes, build
complex nanostructures, silence disease genes, or even perform
calculations. A new, unnatural, base pair could multiply and diversify
these applications.

The most challenging goal, says Romesberg, will be to incorporate
unnatural base pairs into the genetic code of organisms. "We want to
import these into a cell, study RNA trafficking, and in the longest
term, expand the genetic code and 'evolvability' of an organism."

Stanford University chemist Eric Kool, has studied the fundamental
chemistry of base-pair bonding. He foresees challenges, but great
potential in the unnatural bases.

"It requires a long effort by multiple laboratories, but I think
ultimately it will lead to some important tools," he says. "The ability
to encode amino acids with unnatural base pairs will be quite powerful
when it comes."

Journal reference: Journal of the American Chemical Society (DOI:
10.1021/ja078223d)

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