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November 27, 2005
Biotechnology's Newest Chemical Tool
Exploiting biology's own chemical toolbox, researchers have
developed a new technique that will allow them to modify specific
sequences within a DNA molecule. The approach will not only help reveal
the impact of biochemical alterations to DNA, but could have
far-reaching implications for DNA-based medical diagnosis and
nanobiotechnology.
Combining chemistry with biotechnology, Saulius Klimasauskas, a
Howard Hughes Medical Institute (HHMI) international research scholar
at the Institute of Biotechnology in Vilnius, Lithuania, and chemists
at the Institute of Organic Chemistry in Aachen, Germany, have
harnessed a group of essential enzymes to add various chemical groups
to DNA, thereby altering its function. The work was published in an
early online publication on November 27, 2005 in Nature Chemical
Biology.
“DNA methyltransferases will become a standard laboratory tool like restriction endonucleases.”
Saulius Klimašauskas
The enzymes at the heart of the study, known as DNA
methyltransferases, are one of the tools cells use to turn genes on and
off. By adding a simple cluster of four atoms — a carbon atom
attached to three hydrogens, known to chemists as a methyl group
— to specific bases within a DNA sequence, methyltransferases can
effectively shut a gene off. Methylation plays an important role in
embryonic development, genomic imprinting, and carcinogenesis because
it regulates gene expression.
 | | | | |  | | | | | |  |  | Methyltransferase-assisted transfer of extended groups onto DNA
Simulated model of the HhaI methyltransferase... more
Image: Grazvydas Lukinavicius
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Methyltransferases require a source for the methyl groups that they
attach to DNA, and most often that source is a molecule called
S-Adenosyl-L-methionine (AdoMet), sometimes known as SAM or SAMe.
Methyltransferases grab the methyl group from AdoMet and transfer it
directly to DNA, positioning it with enviable specificity within the
sequence. This specificity suggests that the enzymes can be a useful
tool in the laboratory. But Klimasauskas and colleagues wanted the
flexibility to attach more than just a simple methyl group.
In this study, the scientists demonstrated that methyltransferases
can indeed be used to transfer larger chemical groups to large DNA
molecules, in the same sequence-specific manner.
To try out their technique, the scientists synthesized molecules
that mimicked AdoMet, but had chemical groups with longer carbon chains
in the position where the methyl group was usually located. The enzymes
were able to grab the bulkier group and transfer it to DNA. Since the
family of DNA methyltransferases includes enzymes capable of
recognizing more than 200 distinct sequences, this new approach
provided an unprecedented ability to manipulate DNA experimentally.
To demonstrate the technique's potential to alter DNA function, the
researchers modified DNA in a position that blocked another enzyme's
ability to snip the molecule at its target site. “No one has
really thought about possible applications [of this] before because no
one thought it was possible,“ said Klimasauskas. He predicts that
DNA methyltranferases will become a standard laboratory tool like
restriction endonucleases.
Earlier studies had suggested that the transfer of chemical groups
larger than a methyl group would not be possible, in part because
replacing AdoMet's methyl group lowered the chemical reactivity of the
compound. To overcome this problem, the authors took some tips from
organic chemistry textbooks and stabilized the transfer with a multiple
carbon bond.
“It turned out that our first bet, a double or triple
carbon-carbon bond, placed next to the transferable carbon unit, helped
to alleviate the problems that had plagued the reaction in previous
studies,” Klimasauskas said. He likened the chemical reaction to
a mechanical spring, explaining that the chemical energy trapped in
AdoMet is sufficient to deliver a small methyl group to its target
compound. But delivering a larger compound required an auxiliary
"spring" to ensure it would reach the target. So, he said,
“chemical thinking" helped resolve the problematic enzymatic
reaction.
“By demonstrating the transfer of carbon chains as long as 4
to 5 units, we provide proof of principle that further extensions
should also be tolerated,” Klimasauskas said.
Due to their sequence-specific nature, the scientists found that
methyltransferases have a distinct advantage over other commonly used
labeling techniques for DNA and other biopolymers. “Our approach
allows labeling of large native DNA molecules at specific internal or
terminal loci,” Klimasauskas explained.
While potential applications are many, the researchers next plan to
synthesize new AdoMet analogs to expand the collection of chemical
groups that can be transferred to DNA by methyltrasferases.
Klimasauskas's group is currently working to append useful functional
groups to extended chains. For example, researchers often label
cellular components with a molecule called biotin, because it binds
tightly to another molecule, streptavidin, and thus streptavadin can be
used to retrieve the molecule of interest. If biotin were built into an
AdoMet analog, Klimasauskas said, it could then be used as a molecular
hook to fish out all molecules that would naturally be methylated in
the cell. “There is no comparable way for global analysis of the
methylation targets in the cell,” Klimasauskas observed.
DNA is not the only molecule that is naturally methylated in the
cell — RNA and proteins also undergo methylation, and the enzymes
that carry out these reactions also rely on AdoMet as their methyl
source. Since the chemistry is the same, this technique is likely to be
applicable to those biomolecules as well, further expanding its
utility. Klimasauskas said that one potential application might be to
label various sites in the ribosome — the RNA-based site of
protein production — with bright fluorophores using appropriate
RNA methyltransferases, enabling real-time dynamic studies of the
complicated mechanism of protein translation.
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Versión en español
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Cindy Fox Aisen
