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Arrays of Oligo Analogues PNA and LNA Promise Improved Sensitivity, Specificity

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DNA MAY be a great information storage molecule, but many feel it needs some fine-tuning when it comes to microarrays. Peptide Nucleic Acid (PNA) and Locked Nucleic Acid (LNA) chips might provide one answer.

These DNA mimics promise arrays with greater sensitivity, specificity, or ease of use and could find applications in diagnostics, drug development, and as monitoring devices.

But, as if PNA microarrays were the new Holy Grail, companies interested in the technology — including Applied Biosystems and Samsung — are unwilling to discuss their plans.

Why all the secrecy? PNA arrays are attractive for new players in the field, said Jörg Hoheisel from the German Cancer Research Institute in Heidelberg, because “many of the patents [that cover DNA microarrays] do not necessarily cover the PNA work.”

This is not to say that PNA is unprotected: Applied Biosystems, through its purchase of Boston Probes in November, acquired some key patents for PNA technology.

Also, the cost of PNA monomers is high, although it would likely drop if the market were to expand.

PNA, which resembles DNA in structure but has a peptide instead of a sugar phosphate backbone, has several advantages over DNA arrays. Target DNA or RNA that has bound to a PNA array can be detected directly by mass spectrometry, owing to its phosphates, without prior labeling or amplification. This detection is more sensitive than fluorescent detection methods by several orders of magnitude, Hoheisel said, and could become as user-friendly as optical scanners once mass spectrometers have been adapted. And even though hybridization with PNA is slower than for DNA arrays, there are no cumbersome preparation steps.

Moreover, PNA/DNA duplexes are more stable than a pure DNA duplex, so shorter oligonucleotides can be used. This is expected to increase specificity, although Hoheisel did not think it would make a great difference in practice.  

PNA DIAGNOSTICS?

 Because of their potential ease of use, Hoheisel sees the main application for PNA arrays in “routine applications” rather than research. “I think diagnostics are an excellent opportunity for PNA arrays,” agreed James Coull, director of biochemical sciences at Applied Biosystems and former vice president of R&D at Boston Probes. He added that SNP assays and others that require high specificity would offer the greatest potential. But applications outside the medical arena are also feasible, said Hoheisel — for example in monitoring engineering processes. He has tested short PNA oligos synthesized by standard peptide chemistry in high-density spot arrays for genotyping bacteria, breast cancer polymorphisms “and a few other things.”  

Locking in market share 

With LNA, there is less secrecy than with PNA, perhaps because there is less competition. Danish company Exiqon has jumped ahead of the pack to develop LNA arrays and has built a substantial IP portfolio in the LNA arena.

LNA, which looks like RNA but has a methylene linkage between the 2’-oxygen and the 4’-carbon that restricts the molecule’s conformation, has high affinity for DNA, allowing the use of short oligomers on an array.

Moreover, “LNA is so far the DNA analogue with the highest discriminative power of all,” claimed Lars Kongsbak, vice president of business development at Exiqon. LNA can be made on a standard DNA synthesizer and, as a true DNA analogue, is “fully compatible with molecular biology technologies,” he added.

Exiqon is in control of all the patents covering LNA, Kongsbak said, and has licensed them to several companies. But as with PNA, LNA is still very costly to make.

Kongsbak sees the main application for LNA arrays in diagnostics, owing to their high specificity. “When you talk about diagnostic tools, specificity is the key issue,” he said, citing SNP detection, expression profiling, detection of alternatively spliced messenger RNA, and comparative genome hybridization, as possible uses.

Exiqon currently offers custom-made LNA spot arrays with up to about 400 features, where the LNA is immobilized on a polymer surface by a photoreaction. Customers include diagnostic and pharmaceutical companies as well as several research labs. But the company is currently looking for a partner to market an array with about 300 capture probes for toxicity studies in C. elegans, Kongsbak said, and to develop further products.

“For both PNA and LNA the question is whether the advantages they might confer justify the investment in moving from protocols that are already adequate,” commented David Corey, a professor at the University of Texas Southwestern Medical Center at Dallas. Although users will be unfamiliar with some of the new molecules’ properties, he said,“I am optimistic that the answer is ‘yes.’”

— JK

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