By Randall Osborne

By using triplex molecules and a modifying enzyme, CyGene Inc. said it has found a way to solve the "needle-in-a-haystack" problem of finding disease-related genes in DNA and RNA samples.

"We burn the haystack," said Martin Munzer, vice president of Alachua, Fla.-based CyGene, founded two years ago.

The patented technology, called target protection assay (TPA), works on any gene that has been sequenced, handles large samples of DNA and RNA, and gets results fast — detecting, for example, HIV within a week of infection, rather than six or seven months afterward, Munzer said.

"Basically, we can test all the blood at one time," Munzer added.

Leukophoresis, a procedure similar to dialysis, is used to remove white blood cells from a patient for testing.

"We can go up to probably 10 mg of DNA processed at one time, and a human being only has 3 mg of DNA in peripheral blood," Munzer said. "It's almost impossible to find the 'needle' when you're testing just a drop or two at a time."

Vivek Shenoy, CyGene's vice president of science and technology transfer, said using triplex molecules is "not something too many people know about. It's an obscure part of DNA chemistry."

The triplex molecule is formed by synthesizing a small piece of DNA and hybridizing it with the target DNA. Natural DNA, with two strands, is a duplex; the third, synthesized piece of oligonucleotide makes the triplex when it finds the strand it's made to match up with, Shenoy explained.

"After you form the triplex, you throw in an enzyme called exonuclease III," Shenoy said, and the enzyme dissolves or "burns up" one strand of DNA.

"It's very predictable," Shenoy observed. "It only goes after double-stranded DNA, and once it encounters the triplex, it jumps off."

This leaves a triplex-protected target with two long tails. "You know how long the tails are, because you know the size of the fragment you cut out," Shenoy said.

Next, a probe engineered to one of the tails is added, and hybridizes to that region. The TPA technology then magnetically attracts the antibodies attached to the probe, which in turn is attached to the triplex-protected target.

"Then you wash off everything," leaving the protected target, Shenoy said.

Finally, another probe, called a reporter probe, is attached to the second tail of DNA, which is free. At this point the protected target can be amplified.

Munzer said TPA and a similar technology, called restriction fragment target assay (RFTA), have applications across a wide range of diseases — HIV, cancer, hepatitis and more — and could be useful in testing donated organs. "You can extract 80 to 90 percent of the DNA out of [donors'] lymph nodes," which could be tested while the organ is being shipped, he said.

CyGene rents space at the Sid Martin Biotechnology Institute, which is part of the University of Florida, in Gainesville. The company's majority shareholder owns TPA and RFTA.

A second company, Agenda Inc., also of Alachua, is located across the street. It was created to market RFTA exclusively with CyGene's TPA. A patent is pending on RFTA.

"We didn't want [the products] competing with each other, so we decided to do a co-licensing agreement with the two products," Munzer said. "If somebody wants to license one, they have to take both."

Munzer declined to be specific about RFTA, other than to explain that it backs up TPA's results with slightly different methods.

"We can get results in two to four hours with RFTA, and the next day confirm the result on the TPA side," Munzer said. "Both could be automated in one unit. If you were looking for parasites, for example, you would extract the DNA and put it into the device, which would inject the reagents."

The entire procedure would involve "six or eight steps," he said.

While Agenda handles marketing, CyGene will continue with research and development, and search for collaborators, Munzer said. "Eventually, we will merge the two companies," he added.

Shenoy said TPA and RFTA could work well with PCR technology, exclusive rights to which are held by Hoffman-La Roche Ltd., of Basel, Switzerland.

"We would deliver the 'needles' and PCR would then quantify how many needles are present or bring them up by amplification to see whether the target was present or not," he said.

Potential Partners Include DNA Chip Makers

The TPA and RFTA assay methods provide about seven levels of specificity, as compared with PCR's two, Shenoy said. In PCR, the DNA is denatured by heat, and one probe is used for each strand. "If one of those probes ends up being non-specific, you end up amplifying it," he said.

"All of the chip-based technologies are limited to taking pinches of the 'haystack,'" he added, noting that companies using chips — such as Affymetrix Inc., of Santa Clara, Calif. — would be sought as partners.

Using TPA and RFTA, "we can process the DNA and feed it to the chip," Shenoy said.

Among the other companies that could benefit from CyGene's technology, Munzer said, are Nanogen Inc., of San Diego, which works with micro-fabrication of testing equipment; and Perkin-Elmer Corp., of Norwalk, Conn., which manufactures analytical systems used in PCR.

Some companies are showing interest, but no deals have been signed, Munzer said.

"The big boys want it all, and they want it now, and they want it cheap," he said. CyGene is looking for licensees, and wants to continue developing the technologies, Munzer said. *

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