ReviewPeptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology
Introduction
It was clear, even on the introduction of peptide nucleic acids (PNAs) in 1991 [1], that this DNA mimic (see Fig. 1) could play a role in improving existing and developing novel techniques within DNA hybridisation-based methods in genetic diagnostics and molecular biology. It often has been argued that PNA hybridises more strongly to complementary DNA and RNA than do natural deoxyribonucleotides, but with better sequence discrimination; therefore, any hybridisation-based technique employing PNA probes instead of DNA probes should perform better. This is not correct for several reasons. First, the hybridisation strength— expressed as the thermal stability (Tm) for PNA–DNA (and presumably also PNA–RNA) duplexes — display a much more complex sequence dependence compared with DNA–DNA duplexes. A PNA–DNA duplex is not symmetrical and the Tm shows extreme variation that is dependent on the purine content of the PNA strand [2]. This effect is additional to the sequence dependence observed for the stability of DNA–DNA duplexes, and an empirical formula for calculating the Tm of a PNA–DNA duplex (+/–5°C has been derived [3] and is shown in Equation 1:
Where TmDNA is the predicted Tm value for the analogous DNA–DNA duplex according to SantaLucia et al. [4] (not including end effects), and fpyr is the pyrimidine content (or fraction) of the PNA strand.
Notably, these data indicate that at physiological ionic strength, mixed-sequence PNA–DNA duplexes are, in general, slightly more stable (ca.1°C/bp) than the corresponding DNA–DNA duplexes. However, pyrimidine-rich PNA–DNA duplexes are less stable, and purine-rich PNA–DNA duplexes are much more stable than their DNA–DNA counterparts. Furthermore, homopyrimidine PNAs are exceptional in that they form extremely stable PNA–DNA triplexes containing two PNA strands. Therefore, careful sequence considerations are mandatory when designing a PNA probe for use in a hybridisation experiment.
Second, it should be kept in mind that PNA is a noncharged pseudopeptide that has physico-chemical properties that differ significantly from polyanionic oligonucleotides. Therefore, any experimental conditions in hybridisation or other assays that have been optimised for oligonucleotides cannot be expected to be optimal, or even suitable, when employing PNAs.
It also is important to note that the stability of PNA–DNA (or PNA–RNA) complexes (which is due to the noncharged PNA backbone) is virtually insensitive to ionic strength [5], [6]. This contrasts with the stability of DNA–DNA or RNA–RNA duplexes, which are significantly destabilised at very low ionic strength. This property of PNAs can be exploited when targeting DNA or RNA sequences that are involved in or have a propensity to form a secondary structure [7].
Targeting of double-stranded (ds) DNA with PNA can occur via at least four different binding modes (Fig. 2). Three of these modes (e.g. triplex formation, duplex invasion and triplex invasion) require homopurine/homopyrimidine DNA targets, whereas double duplex invasion (i.e. exploiting pseudocomplementary PNA oligomers containing 2,4-diaminopurine and 4-thiouracil instead of adenine (A) and thymine (T), respectively [8]) requires targets of at least 50% AT content. For practical applications, the triplex and double duplex invasion complexes with targets of at least 8 bp will have sufficient stability. It is also important to note that the formation of such invasion complexes is very slow at elevated ionic strength (i.e. >50mM Na+/K+) or in the presence of divalent or multivalent cations (e.g. Mg2+ and spermine). Therefore, binding reactions are performed most conveniently in low ionic strength (<10mM) EDTA-containing buffers.
Considering the range of properties of PNAs, it is not surprising that the most successful applications of PNAs are those that purposely or serendipitously take advantage of and exploit the special properties of PNAs that distinguish them from oligonucleotides (see [9], [10], [11], [12] for recent reviews on PNAs and their applications). In this review the recent advances in using PNA and developing PNA-based technologies for genetic diagnostics and molecular biology are discussed.
Section snippets
In situ hybridisation
PNA probes have proven extremely useful for use in a variety of FISH (fluorescence in situ hybridisation) assays, giving very good signal-to-noise ratios and also allowing employment of hybridisation and washing procedures that yield good chromosome images [13], [14], [15], [16], [17], [18], [19]. Of particular interest, telomeric PNA probes are now being used increasingly in cancer and ageing research, and recently, in combination with centromeric DNA probes to study X-ray-induced chromosome
Nucleic acid capture
A few reports have examined the properties of PNAs as both specific [7], [26], [27] as well as general [28] nucleic acid capture probes (for example, for sample preparation), taking advantage, in particular, of the tight complex formation at low ionic strength under which nucleic acid secondary structure is destabilised significantly [7]. Two recent reports [26], [27] analysed in more detail the performance of PNA oligomers compared with DNA oligomers for capture and recovery of 16S ribosomal
Plasmid vector tagging
The extremely stable and highly sequence-specific complexes 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 variety of ligands through a biotin–streptavidin ‘sandwich’ linker [35]. Essentially, this technology allows an almost irreversible, yet noncovalent and therefore almost ‘biologically silent’, labelling of DNA molecules that can be
Duplex DNA targeting
A major step towards general sequence targeting of dsDNA by PNA, as opposed to the homopurine restrictions inherent in the triplex invasion binding mode, was achieved by introducing pseudocomplementary PNAs that bind to duplex DNA targets via the double duplex invasion mode [8] (Fig. 2). Most, if not all, of the techniques developed based on PNA triplex invasion could take advantage of this new binding mode. Moreover, recently it was demonstrated that a rare cleavage technique exploiting
Detection by solution-phase hybridisation
Direct, real-time detection of DNA targets, for example during a polymerase chain reaction (PCR), is possible using DNA oligonucleotides through beacon technology [37]. The beacon technology takes advantage of a partly self-complementary oligo(nucleotide) hybridization probe containing a fluorophore at one end and a quencher at the other end. When the probe is not hybridized to the target, it forms a hairpin that does not fluoresce because of the close proximity of the quencher and the
PCR clamping
Inhibition of PCR amplification of a specific target by ‘PCR clamping’ [42] (by which a PNA oligomer is used to inhibit the amplification of a specific target, e.g., by direct competition with a PCR primer) has been used very successfully to detect and screen for single base-pair gene variants [43], [44]. The technique is so powerful, in fact, that it also can be used to detect single base-pair gene variants on a background of up to a 100-fold excess wild type [45]. This is accomplished most
Conclusions and future perspectives
In the past year, it has become clear that the use of PNAs in in situ hybridisation and PCR clamping are becoming established techniques that have wide and expanding applications. New applications of PNAs also are still emerging, and there is no reason to believe that this trend shall not continue. Many novel chemical modifications of the original aminoethyl glycine PNA backbone have been made and are still being developed [10]. Most of these are not interesting in relation to practical
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
of special interest
of outstanding interest
References (48)
- et al.
Accelerated telomere shortening in the human inactive X chromosome
Am J Hum Genet
(1999) - et al.
Telomere shortening is an in vivo marker of myocyte replication and aging
Am J Pathol
(2000) - et al.
Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water
Mol Cell Probes
(1999) - et al.
Affinity capture and recovery of DNA at femtomolar concentrations with peptide nucleic acid probes
Anal Biochem
(2000) - et al.
Light-up probes: thiazole orange-conjugated peptide nucleic acid for detection of target nucleic acid in homogeneous solution
Anal Biochem
(2000) - et al.
Detection of apolipoprotein B mRNA editing by peptide nucleic acid mediated PCR clamping
Biochem Biophys Res Commun
(1999) - et al.
Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide
Science
(1991) - et al.
Strand displacement binding of a duplex-forming homopurine PNA to a homopyrimidine duplex DNA target
J Am Chem Soc
(1996) - et al.
A formula for thermal stability (Tm) prediction of PNA/DNA duplexes
Nucleic Acids Res
(1998) - et al.
Improved nearest-neighbor parameters for predicting DNA duplex stability
Biochemistry
(1996)
PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules
Nature
Ionic effects on the stability and conformation of peptide nucleic acid complexes
J Am Chem Soc
Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection
BioTechniques
Double duplex invasion by peptide nucleic acid: a general principle for sequence-specific targeting of double-stranded DNA
Proc Natl Acad Sci USA
Peptide nucleic acids. Analogs and derivates
Curr Org Chem
Antisense peptide nucleic acids
Curr Opin Mol Ther
Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future
FASEB J
Heterogeneity in telomere length of human chromosomes
Hum Mol Genet
Improved in situ detection method for telomeric tandem repeats in metaphase spreads and interphase nuclei
Mol Pathol
Combined fish with pan-telomeric PNA and whole chromosome-specific DNA probes to detect complete and incomplete chromosomal exchanges in human lymphocytes
Int J Radiat Biol
Discrimination between complete and incomplete chromosome exchanges in X-irradiated human lymphocytes using FISH with pan-centromeric and chromosome specific DNA probes in combination with telomeric PNA probe
Int J Radiat Biol
Analysis of radiation-induced chromosomal aberrations using telomeric and centromeric PNA probes
Int J Radiat Biol
Unique chromosome identification and sequence-specific structural analysis with short PNA oligomers
Mamm Genome
Cited by (212)
The role of Nucleic Acid Mimics (NAMs) on FISH-based techniques and applications for microbial detection
2022, Microbiological ResearchCitation Excerpt :Due to their chemical structure, PNA has shown to have many advantages compared with traditional DNA probes. Because PNA probes are neutrally charged and shorter (15 bp) than DNA probes (20–24 bp) (Kenny et al., 2008; Nielsen, 2001), they can more easily penetrate into cells and have consistent hybridization performance because it presents reduced electrostatic repulsion between the non-charged polyamide backbone and the charged DNA/RNA phosphodiester backbone. This contributes to improved thermal stability of the duplex and therefore hybridization can be performed under low salt concentrations, a condition which favors the destabilization of rRNA secondary structures and results in an improved access to target sequences (Rocha et al., 2016).
Cholesterol Anchors Enable Efficient Binding and Intracellular Uptake of DNA Nanostructures
2019, Bioconjugate ChemistryA Synthetic Methodology Toward Pyrrolo[2,3- b]pyridones for GC Base Pair Recognition
2018, Organic LettersPyrrolidinyl PNA polypyrrole/silver nanofoam electrode as a novel label-free electrochemical miRNA-21 biosensor
2018, Biosensors and BioelectronicsCitation Excerpt :Recently, peptide nucleic acid (PNA) has attracted attention as a probe of choice. PNA is a DNA mimic in which the natural deoxyribose-phosphate backbone is replaced by a neutral pseudopeptide chain (Nielsen, 2001). Its neutral backbone lessens the electrostatic repulsion between PNA and RNA, subsequently enhanced hybridization efficiency (Zhang et al., 2009).
Peptide nucleic acid-zirconium coordination nanoparticles
2023, Scientific Reports