Company probes possibilities for PNA
William Check, PhD
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While current molecular diagnostic methods use DNA or RNA, one company is developing assays that use PNA—peptide nucleic acid—a technology invented in Denmark around 1990.
At that time, Jens Hyldig-Nielsen, PhD, was working on DNA-based hybridization methods at Dako. "We did a prestudy in collaboration with the company founded by the inventors of PNA," Dr. Hyldig-Nielsen says. The results led Dako to take a license on PNA probes for diagnostic applications. (PNA is sufficiently different from DNA to be outside the extensive patents that restrict the use of DNA technology.) Dako set up Boston Probes Inc., in Bedford, Mass., to develop PNA probes.
Dr. Hyldig-Nielsen, now director of molecular biology at Boston Probes, explains that PNA was initially devised as a therapeutic reagent. Naked DNA is quickly broken down in the body. To inhibit degradation, a molecular chimera was formed: The sugar-phosphate backbone of DNA was removed and a polyamide backbone, like that in peptides, was inserted. In PNA, the bases are attached to this peptide backbone. (For a display, go to the Web site www.bostonprobes.com. Click on "The Science" and open "PNA Presentation.")
In diagnostics, PNA’s advantage is that no negative charges are carried on its peptide backbone. In DNA, the phosphate backbone is negatively charged, so DNA molecules repel each other. For successful hybridization, this repulsion must be overcome, most commonly by using high salt concentrations. Because of its neutral backbone, PNA hybridizes faster and with higher affinity to RNA and DNA than RNA and DNA hybridize to themselves. And PNA hybridizes under low-salt conditions that destabilize natural hybridizations between RNA and DNA.
"This has a tremendous influence on the efficiency with which PNA hybridizes to highly structured targets like rRNA," Dr. Hyldig-Nielsen says. Because rRNA is present in high amounts, it is a very attractive target for diagnostic detection. The problem with DNA probes is that, under conditions where DNA can hybridize, rRNA tends to form its secondary structure. "As a result, many areas of rRNA are, in practice, off limits for DNA probes," Dr. Hyldig-Nielsen says. "Some of those areas are interesting because they contain important sequence variations."
Boston Probes is marketing bacterial probes, primarily for research use, against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, all enteric salmonella, a universal bacterial probe, and a universal eukaryotic probe.
Boston Probes is collaborating with the Cleveland Clinic Foundation to evaluate a PNA probe for clinical use to confirm S. aureus in blood cultures. "With practically 100 percent efficiency, this probe defined whether S. aureus was present as early as two to three hours," Dr. Hyldig-Nielsen says. Confirmation is always needed when testing human blood samples, he adds. "Confirming S. aureus on selective medium could take one to two days."
A collaboration with a German laboratory is underway to evaluate another of Boston Probes’ PNA probes to distinguish Candida albicans from Candida dublinienis in culture samples.
Boston Probes is also pursuing direct detection in clinical specimens. "Our probes against rRNA, which is present in thousands of copies, give very bright signals," says Dr. Hyldig-Nielsen.