Photochemical technique recombines DNA into various shapes
David L. Shenkenberg
The traditional enzymatic method of connecting DNA molecules cannot assemble them into shapes such as branched or R-shaped DNA, but a new photochemical method can. It promises to become a valuable gene manipulation technique if further improvements can allow it to be used with standard biotechnological procedures.
Kenzo Fujimoto and Masayuki Ogino of the Japan Advanced Institute of Science and Technology in Ishikawa developed the photochemical method. It requires a DNA strand that contains the photochemical on either end, a template DNA strand, and the two DNA strands that will be connected. The template directs the way that the DNA molecules link together, and 366-nm radiation causes the DNA molecules to assemble.
Previously, the scientists connected DNA modified with a photochemical to unmodified DNA. They synthesized both the modified and unmodified DNA and did not obtain either from an organism.
This time, they tested whether the method can assemble two unmodified DNA molecules into the shape of the letter “R” because that would suggest that the photochemical method can recombine two natural DNA molecules into complex structures. They believe that the ability to join two natural DNA molecules will make the photochemical method more useful as a biotechnological technique.
Using the template-directed photochemical method outlined above, researchers linked two natural DNA molecules to form R-shaped DNA with a 90 percent yield. This method has advantages over traditional procedures that may lead to its acceptance as a standard gene manipulation technique. Reprinted with permission from Angewandte Chemie.
The investigators used the photochemicals 5-cyanovinyl-1’-α-2’-deoxyuridine and 5-cyanovinyl-1’-β-2’- deoxyuridine. Fujimoto said that the photo- chemicals and DNA strands can be made easily and that no additional reagents are required. Unlike enzymatic methods, the photoreactions do not have stringent temperature or pH requirements, nor do they need a metal cation, which can interfere with other biotechnology methods and reactions.
To irradiate the DNA, the researchers used a 25-W transilluminator from Tokyo-based Funakoshi Co. Ltd. and a 300-W monochromator made by Jasco Inc. of Easton, Md. They monitored the photoreaction with a combined UV-VIS spectrometer and capillary gel electrophoresis system from Beckman-Coulter Inc. of Fullerton, Calif.
To confirm that they had joined the DNA, they used high-performance liquid chromatography to analyze enzymatically digested DNA fragments and employed a different Beckman-Coulter UV-VIS spectrometer for the analysis. For additional validation, they used a mass spectrometer from Applied Biosystems of Foster, Calif.
The technique produced R-shaped DNA with a 90 percent yield, showing that the experiment worked. Fujimoto said that the photoreactions reliably occur with a high yield, as happened in this case. The investigators noted that their photochemical method also may permit DNA processing in a manner that resembles messenger RNA processing. Because previous reports by his lab have shown that 312-nm irradiation can revert DNA structures to their original form, Fujimoto suspects that the R-shaped DNA also can be used as a caged compound.
The researchers plan to create sets of linked pairs of various natural DNA molecules, including pairs of R-shaped molecules. They also will try to integrate their technique with polymerase chain reaction (PCR) amplification. They are experimenting with DNA polymerases -- enzymes used in PCR -- that can accept the photoreactively joined DNA.
Angewandte Chemie International Edition, Nov. 6, 2006, pp. 7223-7226.
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