Lab-based DNA sequence changer tool developed
them to change sequences within a DNA molecule. The tool is set to
have positive implications in DNA lab-based medical diagnosis and
nanobiotechnology.
In the study, the scientists demonstrated that methyltransferases could be used to transfer larger chemical groups to large DNA molecules, in the same sequence-specific manner. The researchers predict that DNA methyltranferases could become a standard laboratory tool like restriction endonucleases.
The research involved scientists synthesising molecules that mimicked S-Adenosyl-L-methionine (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 recognising more than 200 distinct sequences, this new approach provided an ability to manipulate DNA experimentally.
The researchers then modified DNA in a position that blocked another enzyme's ability to snip the molecule at its target site.
Earlier studies had suggested that the transfer of chemical groups larger than a methyl group would not be possible, because replacing AdoMet's methyl group lowered the chemical reactivity of the compound.
To overcome this problem, the authors took stabilised the transfer with a multiple carbon bond.
"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," said Saulius Klimasauskas, a Howard Hughes Medical Institute (HHMI) international research scholar at the Institute of Biotechnology in Vilnius, Lithuania.
"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 added.
Methyltransferases have a distinct advantage over other commonly used labelling techniques for DNA and other biopolymers. This new approach allows labelling of large native DNA molecules at specific internal or terminal loci.
The researchers plan to synthesise new AdoMet analogs to expand the collection of chemical groups that can be transferred to DNA by methyltrasferases.
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 added 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.
The work was published in an early online publication on November 27, 2005 in Nature Chemical Biology.
