16 Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology Peter E Nielsen During the past ten years, the DNA mimic peptide nucleic acid Figure 1 has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and B characterisation of nucleic acids. Most of these methods have B B taken advantage of the very favourable DNA and RNA O hybridisation properties of peptide nucleic acids combined with O– O O the unique properties and opportunities offered by peptide O O O– P O O chemistry. Within the past year, significant progress in in situ P (5′) O O O hybridisation technology has been achieved, which has resulted, O in particular, in reliable and sensitive methods for detection of DNA bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction B B clamping method have been expanded, and novel ways of B exploiting complexes of peptide nucleic acids with double- O O O stranded DNA, such as double duplex invasion complexes and N N N NH PD loops, have been developed. NH NH NH O Address (Amino-terminal) O O Center for Biomolecular Recognition, Department for Biochemistry PNA and Genetics, Laboratory B, The Panum Institute, Blegdamsvej 3c, DK-2200 N Copenhagen, Denmark Current Opinion in Biotechnology Current Opinion in Biotechnology 2001, 12:16–20 Chemical structures of PNA and DNA. 0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. × Tmpred = 20.79 + 0.83 TmDNA Abbreviations –26.13 × f + 0.44 × length (1) ds double-stranded pyr PNA peptide nucleic acid Tm thermal stability Where TmDNA is the predicted Tm value for the analo- gous DNA–DNA duplex according to SantaLucia et al. [4] (not including end effects), and fpyr is the pyrimidine con- Introduction tent (or fraction) of the PNA strand. It was clear, even on the introduction of peptide nucleic acids (PNAs) in 1991 [1], that this DNA mimic (see Figure 1) Notably, these data indicate that at physiological ionic could play a role in improving existing and developing novel strength, mixed-sequence PNA–DNA duplexes are, in techniques within DNA hybridisation-based methods in general, slightly more stable (ca. 1°C/bp) than the genetic diagnostics and molecular biology. It often has been corresponding DNA–DNA duplexes. However, pyrimi- argued that PNA hybridises more strongly to complementary dine-rich PNA–DNA duplexes are less stable, and DNA and RNA than do natural deoxyribonucleotides, but purine-rich PNA–DNA duplexes are much more stable with better sequence discrimination; therefore, any hybridi- than their DNA–DNA counterparts. Furthermore, sation-based technique employing PNA probes instead of homopyrimidine PNAs are exceptional in that they form DNA probes should perform better. This is not correct for extremely stable PNA–DNA triplexes containing two several reasons. First, the hybridisation strength — PNA strands. Therefore, careful sequence considera- expressed as the thermal stability (Tm) for PNA–DNA (and tions are mandatory when designing a PNA probe for use presumably also PNA–RNA) duplexes — display a much in a hybridisation experiment. more complex sequence dependence compared with DNA–DNA duplexes. A PNA–DNA duplex is not symmet- Second, it should be kept in mind that PNA is a non- rical and the Tm shows extreme variation that is dependent charged pseudopeptide that has physico-chemical on the purine content of the PNA strand [2]. This effect is properties that differ significantly from polyanionic additional to the sequence dependence observed for the sta- oligonucleotides. Therefore, any experimental conditions bility of DNA–DNA duplexes, and an empirical formula for in hybridisation or other assays that have been optimised calculating the Tm of a PNA–DNA duplex (+/–5°C has been for oligonucleotides cannot be expected to be optimal, or derived [3] and is shown in Equation 1: even suitable, when employing PNAs. Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology Nielsen 17 Figure 2 Schematic representation of various complexes formed by PNA binding to duplex (a) (b) (c) (d) DNA. The ladder symbolises the DNA double helix, and the PNA oligomer is drawn in bold. (a) Conventional triplex and (b) triplex invasion complexes are formed at homopurine DNA targets with complementary homopyrimidine PNAs. The triplex invasion complexes have extraordinary stability, whereas conventional triplexes have been observed in a few cases and are far less stable than the corresponding triplex invasion complexes. (c) Duplex invasion complexes are formed with some homopurine PNAs are fairly unstable, whereas double duplex invasion complexes (d) are very stable and require modified nucleobase pairs such as Triplex Triplex invasion Duplex invasion Double duplex invasion diaminopurine–thiouracil. Current Opinion in Biotechnology It also is important to note that the stability of PNA–DNA giving very good signal-to-noise ratios and also allowing (or PNA–RNA) complexes (which is due to the non- employment of hybridisation and washing procedures that charged PNA backbone) is virtually insensitive to ionic yield good chromosome images [13–19]. Of particular strength [5,6]. This contrasts with the stability of interest, telomeric PNA probes are now being used DNA–DNA or RNA–RNA duplexes, which are signifi- increasingly in cancer and ageing research, and recently, in cantly destabilised at very low ionic strength. This combination with centromeric DNA probes to study X-ray- property of PNAs can be exploited when targeting DNA or induced chromosome exchange [18,19]. Further RNA sequences that are involved in or have a propensity developments have resulted in the identification, so far, of to form a secondary structure [7]. ten chromosome-specific PNA probes (for chromosomes 1, 2, 7, 9, 11, 17, 18, X and Y) which utilise satellite repeat Targeting of double-stranded (ds) DNA with PNA can occur sequences. Such probes may become useful for diagnosis via at least four different binding modes (Figure 2). Three of of chromosomal anomalies [20•]. these modes (e.g. triplex formation, duplex invasion and triplex invasion) require homopurine/homopyrimidine DNA PNA–FISH techniques also were developed recently for targets, whereas double duplex invasion (i.e. exploiting detection and identification of bacteria in medical diagnos- pseudocomplementary PNA oligomers containing 2,4- tics [19–23,24•] and in environmental analysis (for diaminopurine and 4-thiouracil instead of adenine (A) and example, water pollution) [25•]. In these cases, PNA thymine (T), respectively [8••]) requires targets of at least probes specific for sequences of the ribosomal RNA of the 50% AT content. For practical applications, the triplex and microorganism are employed [23,24•] and (quantitative) double duplex invasion complexes with targets of at least 8 bp analysis was performed by fluorescence microscopy or by will have sufficient stability. It is also important to note that laser scanning of filtered bacteria. This technique is very the formation of such invasion complexes is very slow at ele- fast and has good sensitivity, but does not readily distin- vated ionic strength (i.e. >50 mM Na+/K+) or in the presence guish naturally between live and dead bacteria. of divalent or multivalent cations (e.g. Mg2+ and spermine). Therefore, binding reactions are performed most convenient- Nucleic acid capture ly in low ionic strength (<10 mM) EDTA-containing buffers. A few reports have examined the properties of PNAs as both specific [7,26,27] as well as general [28] nucleic acid Considering the range of properties of PNAs, it is not surpris- capture probes (for example, for sample preparation), tak- ing that the most successful applications of PNAs are those ing advantage, in particular, of the tight complex formation that purposely or serendipitously take advantage of and at low ionic strength under which nucleic acid secondary exploit the special properties of PNAs that distinguish them structure is destabilised significantly [7]. Two recent from oligonucleotides (see [9–12] for recent reviews on PNAs reports [26,27] analysed in more detail the performance of and their applications). In this review the recent advances in PNA oligomers compared with DNA oligomers for capture using PNA and developing PNA-based technologies for and recovery of 16S ribosomal DNA from very dilute genetic diagnostics and molecular biology are discussed. (i.e. femtomolar) samples. The results indicated no gener- al advantage to using PNAs, but also (not surprisingly) In situ hybridisation showed that both the absolute and relative performance of PNA probes have proven extremely useful for use in a vari- PNA and DNA capture probes is heavily dependent on the ety of FISH (fluorescence in situ hybridisation) assays, experimental conditions (e.g. ionic strength). These 18 Analytical biotechnology Figure 3 Figure 4 (a) B NH N DNA oligo PNA PNA O Current Opinion in Biotechnology Chemical structure of aep–PNA (N-[2-aminoethyl]propyl–PNA). (b) designed [31•] (Figure 3b,c). Analogous to the pad-lock hybridisation system [32], this principle could be devel- oped for signal-amplified in situ hybridisation techniques. Ligation Plasmid vector tagging The extremely stable and highly sequence-specific com- plexes formed upon triplex invasive binding of homopyrimidine PNAs to dsDNA has been exploited as a means of tagging plasmid DNA vectors with fluorophores [33], targeting peptides [34] and, most recently, with a vari- (c) ety of ligands through a biotin–streptavidin ‘sandwich’ linker [35•]. Essentially, this technology allows an almost irreversible, yet noncovalent and therefore almost ‘biolog- ically silent’, labelling of DNA molecules that can be used Earring complex for transfection and eventually for gene therapy. Current Opinion in Biotechnology Duplex DNA targeting Schematic representation of PD loop. (a) Two homopyrimidine ‘PNA A major step towards general sequence targeting of openers’ binding to closely positioned sites create one large DNA loop dsDNA by PNA, as opposed to the homopurine restric- to which an oligonucleotide can bind.