subject: Peptide Nucleic Acids: Robust Probe Hybridization Technology [print this page] Peptide nucleic acid (PNA ) is an artificially synthesized polymer that is capable of binding DNA and RNA in a sequence-specific manner. Since the discovery of its unique binding properties, PNA has been employed in a wide variety of biomedical applications, including genetic research, diagnostics, and experimental therapeutics (1). This article will focus on the diagnostic PNA assays that have gained widespread use in the pathology setting and briefly touch upon other promising applications of this technology.
Unlike DNA and RNA , which have backbones of repeating sugarphosphate units, the PNA molecule is built upon a pseudo-peptide backbone of N-(2-aminoethyglycine) units linked by peptide bonds, to which purine and pyrimidine bases (the specific base-pairing units of nucleic acids) are linked via methylene carbonyl bonds.
The most common usage for PNA molecules are as probes of complementary nucleic acid sequences. As with other nucleic acid probes, the sequences of bases on PNA probes dictate the specificity of binding to complementary DNA and RNA sequences, but the uncharged PNA backbone confers a key advantage to PNA probes. By eliminating the repulsive electrostatic force between traditional nucleic acid probes and their complementary target strands, the neutral PNA backbone confers increased probe affinity and thermal stability to the probe-target duplex.
Specificity of probe binding is a critical aspect of probe assay design, and the physico-chemical properties of PNA probes offer significant advantages for controlling assay specificity. In assays that use traditional DNA or RNA probes, the selectivity conferred by hydrogen bonding between the complementary base pairs on the probe and target strands is offset by the repulsive ionic forces between the strands negatively charged backbones. Optimization of assay specificity requires a delicate balance between parameters such as hybridization temperature, probe concentration, length, and G-C content, and the concentrations of organic solvents and ions, making the design of a robust assay challenging even for experienced diagnosticians. The higher binding energies of PNA probe-target duplexes contributed by the uncharged PNA backbone offer several practical advantages for diagnostic probe assay development. The higher melting temperatures of PNA -DNA duplexes allow PNA probes to invade and overcome many problematic secondary structures in target sequences, and permit very stringent hybridization and wash conditions to be used to increase binding specificity. The higher binding affinities of PNA probes also permit shorter probe sequences and lower probe concentrations to be used in assays, lowering costs and reducing potential non-specific interactions with assay substrates and biological sample components. Mismatches in PNA-DNA duplexes are more de-stabilizing than in corresponding DNA -DNA duplexes, a characteristic which allows PNA s probes to distinguish single base sequence discrepancies such as point mutations and single nucleotide polymorphisms with higher selectivity than DNA or RNA probes.
Another clear benefit of PNA probe chemistry is its exceptional stability. PNA molecules are highly resistant to both nuclease and protease enzymes, and are stable over a wider pH range than DNA or RNA molecules. Probe stability is especially important in diagnostic settings with potentially high amounts of contaminating enzymes, such as assays of minimally processed biological specimens or point-of-use field applications. PNA s stability can also be used to advantage in the design of simplified, rapid diagnostic tests which incorporate PNA probes with other assay components, such as sample preparation reagents, in order to consolidate and reduce steps in the assay procedure.
The majority of the commercial PNA probe products available today are designed for fluorescent in situ hybridization (FISH) assays.
Dako was an early pioneer in the development of PNA -based tests, and in keeping with its pathology focus is using PNA s to enable novel cancer diagnostics. The first PNA probe diagnostic products on the market were Telomere PNA FISH Kit.
These assays, originally conceived and developed by Peter Lansdorps group at the Terry Fox laboratory of the British Columbia Cancer Reseach Center, use PNA probes to quickly and quantitatively visualize the lengths of the telomeric repeat sequences at the ends of each chromosome (2). The kits can be used to assess telomeres in humans and other vertebrate species using interphase nuclei, metaphase spreads, or flow cytometry preparations. Telomere length has been implicated as a critical regulator of a cells capacity for division, and the PNA telomere assays have proven to be valuable tools for studying the relationship between telomere length and cancer, senescence, and other events that influence genetic longevity.
More recently, PNA s have been incorporated into a line of cancer cytogenetic FISH probes, where they are used to enhance assay performance. Each of the FISH products, which include both the Split Signal and Sub-Deletion Signal categories of FISH probes, consists of two DNA fragments (labeled with green and red fluorophores, respectively) complementary to adjacent chromosomal regions that are susceptible to re-arrangement in hematological cancers.
References
1.Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA , et al. PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson-Crick Hydrogen Bonding Rules. Nature 1993; 365:566-8.
2.Lansdorp P, Verwoerd N, van de Rijke F, Dragowska V, Little MT, Dirks R, et al. Heterogeneity in telomere length of human chromosomes. Hum Mol Genet 1996; 5: 685-91.
3.Forrest GN . PNA FISH: present and future impact on patient management. Expert Rev Mol Diag 2007; 7:231-6.
4.Marketed by AdvanDx Inc.
5.Pellestor F, Paulasova P, Hamamah S. Peptides nucleic acids (PNA s) as diagnostic devices for genetic and cytogenetic analysis. Curr Pharm Des. 2008; 14:2439-44.
6.Karkare S, Bhatnagar D. Promising nucleic acid analogs and mimics: characteristic features and applications of PNA , LNA , and morpholino. Appl Microbiol Biotechnol. 2006 ;71:575-86.
