Designing drugs and knowing exactly how they work may have just gotten easier, thanks to researchers at Georgia Institute of Technology.
Using a scanning force microscope, two chemistry graduate students and their research advisors have developed a new way to examine and quickly map how nucleic acid ligands, in some cases anti-cancer drugs, bind to and alter DNA at the molecular level.
This new method works by imaging individual DNA molecules. It's faster than traditional methods, and the results are direct and relatively simple to interpret.
"Our technique directly visualizes individual DNA molecules, while traditional techniques are indirect and inferential, sometimes giving inexplicable results," said Dr. Loren Williams, an associate professor in Georgia Tech's School of Chemistry and Biochemistry who serves as co-advisor to the project.
"Now when a medicinal chemist synthesizes a potential drug, we can very quickly tell them if it binds to DNA, how tightly it binds, and the mode by which it binds," he added.
One of the major advantages of the new assay is it shows researchers how drugs affect single DNA molecules, said Dr. Lawrence Bottomley, a Georgia Tech chemistry professor who specializes in scanning force microscopy.
"Other techniques require us to examine several billion molecules at a time and then make inferences about the behavior of individual molecules," he said.
Fourth-year graduate student Joseph E. Coury, working under Bottomley's direction, is responsible for much of the development of the new assay. Third-year graduate student Lori McFail-Isom, a student of Williams', prepared highly purified drug-DNA samples for the project and currently is obtaining images of new DNA complexes.
Williams and Bottomley presented this work during the 211th American Chemical Society national meeting in New Orleans in late March, and have submitted it for publication in the Proceedings of the National Academy of Sciences.
Dr. Jonathan B. Chaires of the University of Mississippi Medical Center's Department of Biochemistry, called the work "a significant and important advance in the area of drug-DNA interactions."
"One can imagine that the method could be of wide use in the pharmacology industry as an important preclinical screen of new DNA binding agents," said Chaires, who has studied DNA-drug interaction for nearly 20 years. "Since some of the most effective agents in use in cancer chemotherapy are DNA intercalators, this fundamental research could have important practical use."
Bottomley and Williams believe their approach offers many advantages over traditional techniques. Unequivocal methods like X-ray crystallography and NMR spectroscopy are extremely labor intensive. Crystallography, a field in which Williams specializes, is limited to small fragments of DNA, such as 10 to 30 base pairs.
The scanning force microscopy assay can be performed on long pieces of DNA -- from 300 to over 100,000 base pairs -- that more closely resemble the genetic material in the cells of living organisms.
Traditional methods that derive information from longer DNA strands include linear and circular dichroism, viscometry and sedimentation. These methods are generally reliable when ligands bind by conventional intercalative or minor groove modes, but are unreliable for mixed and non-classical modes.
For this particular project, Coury and McFail-Isom looked at three different DNA ligands: ethidium, daunomycin hydrocloride and 2,5-bis(4-amidinophenyl) furan, or APF. Ethidium and daunomycin were used as controls, since their intercalative modes-of-binding already were well-defined. Ethidium is a common DNA stain, while daunomycin is a major anti-cancer drug used primarily to treat leukemia.
APF offered an ideal test of the new method because it is an experimental drug whose mode-of-binding was ambiguous. First synthesized at Georgia State University, the drug is not yet commercially available but has shown early promise in treating Pneumocystis carinii pneumonia, one of the leading causes of death in AIDS patients.
The assay resolved the ambiguities surrounding APF, showing that it does not intercalate in DNA.
The Georgia Tech researchers have been working in this area for about 1 1/2 years. Sponsors include the American Cancer Society, the Research Corporation and the Analytical Division of the American Chemical Society.
Both Williams and Bottomley anticipate widespread acceptance and utilization of this assay once the school's findings are disseminated.
"This is just the beginning," Bottomley said. "Although scanning force microscopes are already available in numerous academic and industrial labs across the country, they have not been widely used in pharmacological applications. Now these microscopes can play a vital role in the design of new anti- cancer and anti-viral drugs, by providing important information on how drugs bind to genes."
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