Definition and Properties. Peptide nucleic acids (PNAs) constitute a new class of DNA probes, that pro­vide an interesting alternative to FISH and PRINS. The PNAs are synthetic mimics of DNA in which the deoxy­ribose phosphate backbone supporting the nucleic acid bases, is replaced by a non-charged peptide backbone [Fig. 2(A)]. The unique chemical makeup of these probes confer a number of beneficial properties, including en­hanced hybridization rates, resistance to nucleases and proteases, and the ability to penetrate condensed biologi­cal structures. The neutral backbone of PNA provides strong binding between PNA/DNA or PNA/RNA strands and greater specificity of interaction than their DNA coun­terparts. While they hybridize according to normal Watson-Crick base pairing rules, PNAs have been shown to bind to DNA or RNA targets with higher affinity than the corresponding oligonucleotides. Unlike DNA probes, that require high salt concentration to bind, PNA probes can bind to DNA or RNA under low ionic strength condi­tions that disfavor re-annealing of complementary strands. Experiments with homopyrimidine strands have shown that the Tm of a 6 mer PNAT/DNAdA was 31°C in compari­son to a DNAdt/DNAdA 6-mer duplex that denatures at a temperature of less than 10°C. Experiments done with PNA probes containing all four bases have demonstrated that there is an increase of the Tm of about 1°C per base pair in PNA hybrids compared to DNA/DNA or RNA/ RNA duplexes. In addition, a PNA/DNA mismatch is more destabilizing than a mismatch in a DNA/DNA du­plex. A single mismatch in mixed PNA/DNA 15-mer de­creases the Tm by 15°C. In the corresponding DNA/DNA duplex, a single mismatch decreases the Tm by only 11°C. Moreover, the use of even shorter PNA probes can further increase PNA specificity. This advantage is particularly important for hybridization with short probes targeting repetitive sequences, because both the length and the re­petitive nature of these genomic targets will favor re-naturation over hybridization with probes.

These outlined properties mean that it is not necessary to design long PNA probes. Short PNA oligomers, from17 to 22 bases units, constitute efficient tools for detecting specific DNA sequences with fast hybridization kinetics (20 to 60 min.) over a wide pH range. In addition, PNAs are not limited to any detection procedure. The PNAs can be labeled with a large variety of reporter molecules (en­zymes, haptens, fluorophores) [Fig. 2(B)].

Applications and Perspectives. The PNA was origi­nally conceived as reagents for sequence-specific recogni­tion of double-stranded DNA via triple helix formation [26]. However, the unique properties of PNA as a DNA mimic have quickly led to the development of various applications. Most notably, PNAs find current use in mo­lecular biological techniques as specific and sensitive probes for complementary nucleic acids. The PNA oligo­mers are powerful probes in Southern and Northern blot­ting but also for mutation research. Polymerization of PNA oligomers into polyacrylamide gels creates a me­dium in which the unique properties of PNA/DNA interac­tions are utilized to achieve hybridization with single-stranded target DNA during affinity electrophoresis. Such a procedure has been used to identify single point muta­tions [27]. However, the greatest benefit of PNA comes from the development of new diagnostic assays. For exam­ple, PNA technology drastically reduces assay times com­pared to standard methods for bacterial analysis, by using PNA probes that target specific ribosomial RNA sequences [28]. Because of their high affinity and stabil­ity, PNA oligomers are also employed in transcription inhibition (antigene strategy) and translation inhibition (antisens strategy) [29]. The use of PNA probes in cyto­genetics, as an efficient variation to FISH for chromo­somal identification, is recent. Firstly, it has been demon­strated that PNA probes are useful for detecting telomere repeat sequences [30] and triplet repeat sequences in myo­tonic dystrophy cells [31]. The high discriminating ability of PNA probes was evidenced by the detection of inter-spaced centromeric DNA repeats that differed by a single base pair [32], suggesting the further use of PNAs for differentiating chromosomal polymorphisms. The avail­ability of chromosome-specific centomeric PNA probes, directly fluorochrome-labeled, has led to the development of rapid PNA protocols for the in situ detection and enu­meration of human chromosomes in metaphases and inter­phase nuclei. Multicolor PNA experiments were thus re­ported on cells from normal subjects and patients with numerical abnormalities [33,34]. The procedure was also adapted on human sperm [35]. On this material, PNA gave a similar performance to PRINS, pointing out the advan­tages of both these techniques over conventional FISH in terms of hybridization kinetics, specificity and accessibil­ity to the targeted sequences. The successful adaptation of the PNA technique on human sperm has proven that these probes could be used on difficult and compact biological material.