New ways to light up diseases: These yellow peptide nucleic acids could pick out ataxia-telangiectasia, an inherited and disabling neurological disease.
P Is The New D
In most hospitals, patients are treated for infections before their doctors can be sure what's wrong. Diagnostic tests take so long that physicians must simply make their best guess as to appropriate treatment and then alter it if necessary when test results come back—24 to 36 hours later. Prescribing inappropriate antimicrobial drugs risks both subpar patient treatment and the development of drug-resistant pathogens, but there's little that traditional microbiological methods can do to speed up the process. But some hospitals are now using synthetic molecules called peptide nucleic acids, or PNAs, to cut the time needed for diagnosis of common infections to just a couple of hours. "In clinical microbiology, time is key," says Byron Brehm-Stecher of Iowa State University in Ames. "The faster you can make a diagnosis, the faster you can prescribe effective and appropriate antibiotic therapy."
PNAs are also used in many other applications in biotechnology and medicine. These molecules, which are essentially hybrids of deoxyribonucleic acid (DNA) and proteins, are leading to especially promising inroads in anticancer and gene therapeutic drugs.
The PNA story began in 1991 when researchers from the University of Copenhagen published their synthesis of the first PNA molecule. Their idea was to create a molecule that could physically prevent DNA from being transcribed into ribonucleic acid (RNA), thereby blocking expression of faulty genes. The researchers, led by Peter E. Nielsen, attached the familiar adenine, cytosine, guanine and thymine bases of DNA to a backbone made of peptide units similar to those in proteins. (See Nielsen, PE. 2008. A new molecule of life? Scientific American 299(6):64–71.) This combination conferred qualities that made PNAs attractive for some applications that normally employ DNA. For example, DNA backbones carry a negative electrical charge, but peptide backbones are electrically neutral. This difference means that PNA binds much more strongly to DNA than does DNA itself. PNA is also unusually stable inside a cell, because the enzymes that break down normal proteins and nucleic acids don't affect it.
Nielsen and his colleagues found that PNA could indeed prevent transcription of chromosomal DNA by displacing one of the strands of the double helix. But Nielsen and others soon realized that PNA's unusual qualities would lend themselves to an unexpected variety of applications.
Pnas In Microbiology
Soon after PNA's appearance, scientists began synthesizing short stretches of it to use as probes to detect specific DNA or RNA sequences inside cells. Although similar techniques had existed for years with DNA or RNA molecules as probes, PNA's extraordinary stability and hybridization strength made it attractive to use instead.
One of PNA's most successful applications is in detecting and identifying microorganisms. Identifying pathogens quickly is important for treating infections in humans and other animals, as well as for protecting food, beverages and municipal water supplies. In the late 1990s, Henrik Stender, then at Dako A/S in Glostrup, Denmark, addressed such issues by adapting a technique called fluorescence in situ hybridization (FISH) for use with PNA. In traditional FISH, fluorescently labeled DNA probes are sent inside cells, where they bind to complementary DNA or RNA, revealing the presence of the target sequence. The new technique—called PNA-FISH—uses PNA probes to target ribosomal RNA (rRNA), which is found in all organisms, and its sequence is commonly used to distinguish microbial species. Stender and his colleagues designed a PNA probe complementary to the rRNA sequence of the tuberculosis bacterium and found that it reliably differentiated between healthy and infected samples.
In 2003 the U.S. Food and Drug Administration approved three PNA-FISH kits created by AdvanDx, a company that Stender founded in Woburn, Mass. These tests—for Staphylococcus and Enterococcus bacteria plus Candida yeast—are now used at hospitals around the country. A PNA-FISH test to identify a microorganism in a patient sample currently takes hospital microbiologists about two and a half hours—at least 10 times faster than traditional diagnostics. Plus, researchers found that using PNA-FISH to diagnose staph infections in hospitals reduces antibiotic use, total hospitalization costs, length of stay and patient mortality. "Faster results allow physicians to make better decisions regarding therapy," Stender says.
PNA-FISH can also be used to identify environmental contaminants like Listeria and Salmonella in bottled water, on public beaches and in wine and food. "It's broadly applicable to microorganisms in any type of sample," Stender says, although non-clinical applications are not yet commercialized. According to Brehm-Stecher, "For foods and environmental microbiology, PNAs are primarily still in the research phase."
Pushing The Potential Of PNAS
PNA's potential goes far beyond microbiology. In 2007 a group at the University of Oxford reported that PNA can alleviate symptoms of muscular dystrophy in mice. Muscular dystrophy is caused by dysfunction of the protein dystrophin. The researchers injected the mice's muscles with PNA that interfered with expression of a faulty part of the dystrophin gene, which then allowed the mice to make enough normal protein to function.
PNAs also appear to have anticancer potential. A group led by David Corey at the UT Southwestern Medical Center at Dallas showed that PNAs can interfere with the enzyme telomerase, whose activity can lead cells to become cancerous. "By inhibiting telomerase, we were able to slow the growth of tumor cells," Corey says. A molecule that is a direct descendent of this PNA is currently in clinical trials as an anticancer agent.
More recently Corey's group published work that furthers the hope for using PNA as originally intended: to silence genes by binding to chromosomal DNA. Although some technical problems have plagued this technique over the years, Corey and his colleagues recently found that PNA can access chromosomal DNA in living cells, suggesting that PNA might have many more uses ahead, he says. If PNA can bind chromosomal DNA, it might also be able to deliver a chemical reagent that could correct a mutation or recruit proteins that activate gene expression. "All sorts of other applications start to suggest themselves," Corey says.
