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Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future

Department of Physical Chemistry, Chalmers University of Technology, S 412 96, Gothenburg, Sweden

¹Correspondence: Department of Physical Chemistry, Chalmers University of Technology, S 412 96, Gothenburg, Sweden. E-mail: norden@phc.chalmers.se; ray{at}phc.chalmers.se

	ABSTRACT

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic. It is chemically stable and resistant to hydrolytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell. PNA is capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It may also recognize duplex homopurine sequences of DNA to which it binds by strand invasion, forming a stable PNA-DNA–PNA triplex with a looped-out DNA strand. Since its discovery, PNA has attracted major attention at the interface of chemistry and biology because of its interesting chemical, physical, and biological properties and its potential to act as an active component for diagnostic as well as pharmaceutical applications. In vitro studies indicate that PNA could inhibit both transcription and translation of genes to which it has been targeted, which holds promise for its use for antigene and antisense therapy. However, as with other high molecular mass drugs, the delivery of PNA, involving passage through the cell membrane, appears to be a general problem.—Ray, A., Nordén, B. Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future.

Key Words: DNA • PNA-DNA hybridization • antigene • antisense • gene therapy • nucleic acid biosensor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
PEPTIDE NUCLEIC ACIDS (PNA) originated from efforts during the 1980s in organic chemist Prof. Ole Buchardt’s laboratory in Copenhagen together with biochemist Peter Nielsen to develop new nucleic acid sequence-specific reagents. Based on the observation with flow linear dichroism that -helical poly--benzylglutamate (PBG) can form stacked complexes with aromatic chromophores, it was proposed that PBG with alternating nucleobases and acridine moieties instead of phenyls might bind sequence selectively to duplex DNA by combined Hoogsteen base pair formation and intercalation with the helix backbone in the major groove. The proposed peptide nucleobase compound was given the ad interim name peptide nucleic acid, or PNA.

Today’s PNAs (Fig. 1 ) are DNA analogs in which a 2-aminoethyl-glycine linkage generally replaces the normal phosphodiester backbone (1 , 2) . A methyl carbonyl linker connects natural as well as unusual (in some cases) nucleotide bases to this backbone at the amino nitrogens. This simple and yet entirely new synthetic molecule has an interesting and nonprototype chemistry. PNAs are non-ionic, achiral molecules and are not susceptible to hydrolytic (enzymatic) cleavage. Despite all these variations from natural nucleic acids, PNA is still capable of sequence-specific binding to DNA as well as RNA obeying the Watson-Crick hydrogen bonding rules (3 , 4) . Its hybrid complexes exhibit extraordinary thermal stability and display unique ionic strength properties. Although PNA was earlier considered primarily a potential drug candidate for gene therapy, today there are three to four major groups of applications for this novel compound. First, it can be used as a molecular tool in molecular biology and biotechnology (5 6 7 8 9) . Second is its role as lead compound for the development of gene-targeted drugs applying antigene or antisense strategy (10 11 12) . Third is the use of PNA for diagnostics purposes and development of biosensors (13 14 15 16) . Fourth, the study of basic chemistry is related to PNA (17 18 19 20) for the improvement of basic architecture, e.g., for supramolecular constructs and to possibly generate a subsequent generation of PNA molecules.

View larger version (11K): [in this window] [in a new window]   Figure 1. Chemical structure of PNA (left) and DNA (right).

This review will focus on different mechanistic aspects in order to understand the physicochemical and biological properties of PNA and PNA–nucleic acid complexes. We will discuss its mode of action in relation to potential use as an antigene or antisense agent and related developments for gene therapeutic application, detection systems for genetic analysis, and novel tools for nucleic acid manipulation. Finally, the cellular delivery of peptide nucleic acids will be discussed in light of recent progress in this area.

	Chemical properties and related methodology

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
This section will discuss, in brief, the synthesis and purification of peptide nucleic acids. Then the physicochemical properties of PNAs, as characterized from different biophysical measurements, will be discussed.

	Synthesis and purification

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The backbone of PNA carries 2-aminoethyl glycine linkages in place of the regular phosphodiester backbone of DNA (1 , 2) , and the nucleotide bases are connected to this backbone at the amino nitrogens through a methylene carbonyl linker (Fig. 1) . Being achiral, peptide nucleic acids can be synthesized without need of any stereoselective pathway. PNA oligomers can be prepared following standard solid-phase synthesis protocols for peptides (21 , 22) using, for example, a (methylbenzhydryl)amine polystyrene resin as the solid support (23 24 25 26 27) . The scheme for protecting the amino groups of PNA monomers is based on either Boc or Fmoc chemistry (23 24 25) . The postsynthetic modification of PNA uses coupling of a desired group to an introduced lysine or cysteine residue in the PNA (23 , 27) . Amino acids can be coupled during solid-phase synthesis or compounds containing a carboxylic acid group can be attached to the exposed amino-terminal amine group to modify PNA oligomers. A bis-PNA is prepared in a continuous synthesis process by connecting two PNA segments via a flexible linker composed of multiple units of either 8-amino-3,6-dioxaoctanoic acid or 6-aminohexanoic acid (28) . It has been found that changing cytosine to pseudoisocytosine in the Hoogsteen strand abolishes the pH dependence of a DNA·PNA-DNA triplex without affecting the stability of the triplex (28) .

A subsequent generation of PNAs might contain an unusual modification of the backbone or a chimeric architecture. The latter is commonly known as a PNA/DNA chimera, where a PNA oligomer is fused to a DNA oligomer to give rise to new architecture. The synthesis of PNAs with modified backbones is carried out in a similar way. PNA hetero-oligomers comprising normal aminoethylglycine and aminoproline building blocks are prepared using Boc strategy (for details, see ref 29 ). Two principal strategies have been adopted to facilitate the synthesis of PNA/DNA chimeras: block condensation of presynthesized PNA and DNA oligomers in solution, and stepwise, online solid-phase synthesis with suitably protected monomeric building blocks. The second strategy has been successful for the synthesis of chimera. Development of special protecting group strategies is also going on. Koch et al. (30) have reported the synthesis of a PNA pentadecamer carrying a heptapeptide attached to the NH₂ terminus via a tris(8-amino-3,6,-dioxaoctanoic acid) using Boc peptide synthesis. The method made use of traditional PNA synthesis with Boc-benzyloxycarbonyl strategy. The presence of the peptide does not significantly affect the structure of the PNA–DNA duplex. Betts et al. (31) have reported the synthesis of a PNA-peptide-PNA chimera where two pyrimidine PNA nanomers have been linked by a his-gly-ser-ser-gly-his peptide. This construct is capable of forming a stable triple-helical complex with an oligopurine complex (31) .

Postsynthesis procedures involve cleaving PNA oligomers from the solid support by treatment with either anhydrous hydrogenfluoride or trifluoromethanesulfonic acid, followed by high-performance liquid chromatography (HPLC) purification (23 , 28 , 32 , 33) . The crude PNA products can also be cleaved from the solid matrix by treatment with trifluoroacetic acid and m-cresol (4:1) mixture. Then PNA is precipitated by ice-cold diethyl ether and dissolved in 0.1% trifluoroacetic acid solution (23 , 32 , 33) . Further purification and subsequent characterization are done using reverse-phase HPLC, followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (23 , 28 , 32 , 33) .

	Physicochemical properties

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Since peptide nucleic acids and DNA have no functional groups in common except for the nucleobases, the chemical stability of PNA differs significantly from its DNA counterpart. Unlike DNA, which depurinates on treatment with strong acids, peptide nucleic acids are fairly acid stable. However, some chemical instability can derive from the free amino functionality at the NH₂ terminus, where a slow N-acyl transfer of the nucleobase acetic acid or a cleavage of the amino-terminal PNA unit by ring closure can take place, particularly under basic conditions (34) . PNAs are charge-neutral compounds and hence have poor water solubility compared to DNA. Neutral PNA molecules have a tendency to aggregate to a degree that is dependent on the sequence of the oligomer. PNA solubility is also related to the length of the oligomer and purine:pyrimidine ratio (35) . Some recent modifications, including the incorporation of positively charged lysine residues (carboxyl-terminal or backbone modification in place of glycine), have shown improvement as to solubility. Negative charges may also be introduced, especially for PNA–DNA chimeras, which will enhance the water solubility. The extinction coefficients for PNA monomers are not as well established as they are for DNA or RNA monomers. It is expected that PNA oligomers should have extinction coefficients different from their DNA or RNA counterparts, as dissimilar backbones would variously perturb the system of the nucleobases. Since the extinction coefficients of different PNA monomers are not well characterized and contributions of the peptide backbone to the perturbation in the system of the nucleobases are not known, for all practical purposes the concentration of a PNA oligomer is determined by measuring the absorption at 260 nm at 80°C (36 , 37) . At that temperature, the nucleobases are considered completely destacked and contributions from an ordered backbone can be assumed to be less important.

PNAs hybridize to complementary DNA and RNA sequences in a sequence-dependent manner, following the Watson-Crick hydrogen bonding scheme A, multi-stranded triplex structure containing both Watson-Crick and Hoogsteen base-pairing is also possible from a 2:1 PNA–DNA complex (38 39 40 41) (Fig. 2 ). PNA-DNA hybridization is severely affected by base mismatches and PNA can maintain sequence discrimination up to the level of a single mismatch. On the other hand, base mismatches are less effective for corresponding DNA-DNA hybridization. Different properties as well as structures of different PNAs and PNA–DNA complexes have been well characterized by a combination of various spectroscopic, crystallographic, and computational methods.

View larger version (13K): [in this window] [in a new window]   Figure 2. Schematic representation of different molecular complexes formed on target-specific binding of PNA to DNA. A) Formation of a prototype triple helical structure only possible with cytosine-rich homopyrimidine PNAs. B) Stable triplex invasion complex formed by homopyrimidine PNAs binding to a homopurine DNA target. C) Double-duplex invasion complex only possible with PNAs containing nonstandard nucleobases (116) .

	PNA–DNA/RNA and PNA–PNA duplexes

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The properties of various duplexes of PNA, both pure and hybrid, have been investigated extensively by absorption spectroscopy. The thermal melting temperature (T_m), defined as the temperature at which 50% of the complexes have been dissociated, gives an idea of the stability of the PNA-DNA or PNA–RNA duplex. Moreover, it provides information about different structural transitions. The PNA oligomer H-TGTACGTCACAACTA-NH₂ can form an antiparallel duplex with complementary DNA sequence, which has a T_m value of 70°C. But the corresponding DNA–DNA complex melts at only 53°C. However, the corresponding parallel PNA–DNA duplex shows a transition temperature of 56°C (4) . The thermal stability of a PNA–RNA duplex is even higher than that of a PNA–DNA duplex (42) . In contrast to the pure DNA duplex, the stability of PNA–DNA hybrids is not affected much by changes in ionic strength except in the limit of low ionic strength, where the stability increases. The binding of PNA to a corresponding complementary DNA oligomer takes place in a sequence-specific manner, which means that the thermal stability of a heteroduplex is considerably lowered by the presence of mismatches. Corresponding DNA duplexes are not affected by mismatches in this way. Owing to the higher sequence specificity of PNA on binding to nucleic acids, incorporation of any mismatch in the duplex considerably affects the thermal melting temperature of the heteroduplex. For the heteroduplex comprising the PNA oligomer H-egl-GGCAGTGCCTCACAA-NH₂ (carboxyl-terminal) and its full complementary DNA 5'-TTGTGAGGCACTGCC-3', a mismatched incorporation of A in the in eighth position of the DNA sequence, in place of G, reduces the T_m value from 72.3°C to 58.1°C (Table 1 ; B. Nordén and co-workers, unpublished observations). PNA–DNA chimeras also follow the basic Watson-Crick hydrogen bonding scheme on hybridization with complementary DNA and RNA. The thermal melting temperature of the duplex formed as a result of the interaction between a PNA/DNA chimera and a DNA or RNA counterpart usually lies between that of the corresponding PNA–DNA and DNA–DNA duplexes. The T_m value of the chimera–DNA duplex also depends on the ratio of PNA and DNA in the chimera strand and, above all, on the nature and position of the linker molecule between PNA and DNA bases (ref 29 and references therein).

Apart from these complexes with natural nucleic acids, peptide nucleic acids can also bind to complementary sequences of PNA itself to form extremely stable PNA–PNA duplexes (43) . Preference is found for antiparallel duplexes with high melting temperatures compared to their hybrid (PNA–DNA duplex) counterparts. The PNA decamer H-GTAGATCACT-NH₂ forms both antiparallel and parallel complexes with complementary PNA and DNA sequences. The T_m values corresponding to antiparallel PNA–PNA, PNA—DNA, and DNA–DNA sequences are 67°C, 51°C, and 33.5°C, respectively. However, the parallel PNA–PNA complex with a T_m value of ~47°C is still considerably more stable than the corresponding DNA–DNA duplex (43) . The increased thermal stability of PNA–PNA duplexes relative to the corresponding DNA–DNA duplexes is fundamentally due to the absence of any significant electrostatic repulsion between the two strands in the former complex.

Moreover, a PNA-PNA-PNA triplex construct is also possible from one purine and two pyrimidine decamers, as evidenced from circular dichroic measurements (20 , 44) .

In general, PNA contains an achiral backbone and does not show any circular dichroism (CD). However, PNAs with certain modifications of the backbone structure and its conjugation with complementary DNA and RNA sequences to form hybrids are capable of exhibiting circular dichroic characteristics (Fig. 3 ). A PNA duplex containing L-lysinyl amide attached to the carboxyl terminal of the PNAs can demonstrate a handedness resulting from the helicity of the structure. The chiral orientation of the base pairs relative to each other is regarded as the source of the CD signal, and the measured induced helicity is dependent on the nucleobase sequence proximal center (45) . The CD spectra of DNA–DNA and antiparallel PNA–DNA and PNA–RNA duplexes are more or less similar. This suggests that the base pair geometry of antiparallel right-handed PNA-containing helices is not much different from that found in a B- or an A-form DNA helix. In contrast, the spectra of parallel PNA–DNA and PNA–RNA duplexes deviate more from the DNA-DNA spectrum, which suggests a different kind of base stacking. It has been concluded that incorporation of a PNA strand/strands drastically reduces the binding capability of both minor groove binders and intercalators (46) .

View larger version (22K): [in this window] [in a new window]   Figure 3. CD spectra of GTAGATCACT·AGTGATCTAC duplexes: DNA–DNA (curve 1) and PNA–PNA (curve 2) duplexes. Base pair concentration, 50 µM; path length of the cuvette, 1 cm. Adapted by permission from Dr. Pernilla Wittung.

In solution, the PNA monomer exists as both the cis and trans rotamers about the tertiary amide bond, slightly favoring the trans conformation (20) as revealed from the ¹H-NMR (nuclear magnetic resonance) spectra. The two rotamers interconvert on the time scale of ~1 s at room temperature. For longer sequences, such as the PNA octamer H-GCTATGTC-NH₂, the ¹H-NMR spectrum reveals the existence of a multitude of structural species. For a total of N number of residues, there can be 2^N possible structural species. The solution structures of the antiparallel PNA-DNA (H-GCTATGTC-NH₂5'd[GACATCGC]) duplex have been determined by ¹H-NMR (20 , 47 , 48) . The duplex shows a helical conformation with a Watson Crick base-pairing scheme. The helical rise is ~42 Å with ~13 base pairs per turn, whereas the helical diameter is close to 23 Å. The major groove is wide and deep but the minor groove is quite narrow and shallow. The carbonyl groups on the backbone base linkers of this structure are oriented along the backbone, pointing in the carboxyl-terminal direction. However, the sugar puckering is predominantly in the C2'-endo conformation with all antiglycosidic torsion angles and trans primary amide bond conformations. The PNA–RNA duplex molecule (GAACTC-5'r[GAGUUC]), as revealed from proton NMR spectrum, has features similar to a right-handed A helix (20 , 49) . The carbonyl groups of the PNA backbone linkers are directed toward the carboxyl terminus and primary amide bonds are in the trans configuration. The sugar puckering in the nucleic acid strand is C3'-endo with glycosidic torsional angles near -160°, features similar to an A-type conformation.

Rasmussen et al. (50) reported the crystal structure of a PNA duplex (H-CGTACG-NH₂)₂ at 1.7 Å resolution. The molecule crystallized in space group P_1bar, showing the presence of both a right- and a left-handed helix in the unit cell (50) . The helical parameters estimated for this complex deviate significantly from the canonical A and B conformations. The pitch is large (18 bp) and the helix width is 28 Å, whereas the twist, rise, base tilt, and displacement values are 19.8, 3.2, 1.0, and 8.3 Å, respectively. One helical turn of the PNA duplex is ~58 Å long. From the results of their crystallographic study, Nielsen and co-workers (20 , 50) have confirmed that a sugar phosphate backbone is not a prerequisite for the formation of a nucleobase double helix in accordance with the inferences based on earlier circular dichroism results when this species was discovered (43 , 45) .

It is also pertinent to briefly describe here the thermodynamic aspects of PNA binding to DNA and the binding motif. The need for a detailed thermodynamic characterization of PNA-DNA interaction is essential in order to improve our overall understanding of such a reaction, particularly to accurately predict the thermodynamic properties of relatively new complexes of sequences not studied before. So far there are only a few reports concerning the determination of the thermodynamic parameters H° and S° (4 , 36 , 51) . Two major techniques used to determine thermodynamic parameters are the thermal melting of peptide nucleic acid complexes based on absorption hypochromicity of bases measured at 260 nm and the isothermal titration calorimetry (36 , 51 , 52) . The thermal melting profile or so-called equilibrium melting curve reflects a structural transition from an ordered to a disordered state or vice versa, and thermal melting temperature T_m, H°, and S° are calculated from a van’t Hoff analysis of the curve. In the simplest case, a bimolecular reaction (single strand [PNA] + single strand [PNA/DNA/RNA] duplex) representing a two-state model is assumed for analysis of the melting curves (for a detailed discussion see refs 36 , 51 , 53 54 55 ). The model assumes that each single strand exists in one of two possible states: completely based paired and stacked or completely denatured and unstacked; partial base pairing is not considered. This model holds well for short duplexes (5–20 base pairs). It is important to note that single-stranded peptide nucleic acids, particularly at low temperature, can also exhibit an ordered structure represented by a self-melting profile of the oligomer (36 , 51) .

In contrast to the thermal melting method based on the van’t Hoff principle, calorimetric methods measure the heat of reaction and change heat capacity, C_p, during a reaction; the thermodynamic parameters are determined in a model-independent manner (51 , 52) . Isothermal titration calorimetry (ITC) measures the released or absorbed heat of reaction, at a particular temperature, as a ligand is titrated into a solution containing the reactant (for details, see ref 52 ). Another method, known as differential scanning calorimetry, can also be used to complement the ITC technique. It measures the change in heat capacity directly and the reaction enthalpy, and entropy can be evaluated independent of any model for binding. However, the requirement of a high sample concentration (~100 µM) restricts the use of certain PNAs with low solubility.

The overall free energy (G) can be divided into enthalpic and entropic contributions. As discussed earlier, both PNA–PNA and PNA–DNA complexes are thermally more stable than the corresponding DNA–DNA duplexes. The additional stability of hybrid duplex over a pure DNA duplex can be attributed to a more favorable entropic contribution, whereas the higher stability of a PNA–PNA duplex over its DNA–DNA counterpart is mainly due to the enthalpic contribution (36 , 51) . It is important to mention that for PNA–DNA and DNA–DNA duplexes, enthalpic changes H are rather equal. This is also reflected from the hypochromicity values obtained for both complexes. It has been suggested that the entropic contribution is possibly related to the ion release associated with the formation of a hybrid duplex (36 , 51 , 52) . On the other hand, a higher H value for the PNA–PNA duplex compared to its DNA–DNA counterpart signifies a larger structural rearrangements during the phase transition (from native duplex to coiled state) of the duplex (36 , 51 , 53 , 54 , 55) . A higher hypochromicity value was observed for PNA–PNA duplexes compared to their DNA–DNA counterparts, which is consistent with the above interpretation. At low ionic strength conditions, because PNA backbone is devoid of electrostatic charges, a favorable enthalpy of hybridization is generally observed in this order: PNA-PNA>PNA-DNA>DNA-DNA (36 , 51) . It has been suggested that hydrophobic interactions play an important role for the stabilization of PNA-containing duplexes. The charge-neutral backbone of PNA interacts with the surrounding water molecules, the so-called spine of hydration. The water is probably organized differently in PNA-containing duplexes. The interaction with surrounding water molecules is not as favorable as with a DNA–DNA complex containing charged sugar phosphate backbones, which could possibly explain the relative importance of enthalpic contributions to the free energy of hybridization for the PNA-containing duplexes over DNA–DNA duplexes. It has been observed that the presence of terminal lysine residues contributes to the solubility of a PNA oligomer and stabilizes PNA–DNA duplexes. The H and S values for such lys-PNA–DNA duplexes are less negative than the values determined for corresponding hybrid duplexes containing PNAs without terminal lysine residues. The thermal stability of nucleic acids is strongly dependent on the sequence and so is the thermodynamic information derived from the thermal melting data. The thermal stability range of PNA-containing duplexes has been found to be larger than that for pure DNA duplexes (36 , 51 , 52 , 56) .

	(PNA)2–DNA triplexes

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Homopyrimidine PNAs bind to complementary DNA sequences to form (PNA)₂–DNA triplexes (T_m > 70°C). The stability of the complexes depends on the length of the oligomers in a regular manner with an increase in T_m of ~10°C per base pair. A PNA-(DNA)₂ triplex is observed from an interaction between a C-rich PNA and GC-rich DNA duplex. Since a protonated cytosine is involved in the base triplet formation, this particular triple helical structure involving CGC⁺ triplets shows a pH dependence. The structure is most stable in the pH range of 5.0–5.5. This problem could be circumvented by incorporating pseudo-isocytosines (‘J bases’) in place of normal cytosine, e.g., in bis-PNAs (28) (Fig. 4 ), which can form a C.G*J base triplet instead of C.G*C+, which is essentially independent of pH.

View larger version (16K): [in this window] [in a new window]   Figure 4. Hoogsteen-bonded base triplets (involving pyrimidine*purine·pyrimidine). A) Different base triplets involving Watson-Crick and Hoogsteen hydrogen bonding. B) Schematic representation of the formation of a strand invasion complex involving bis-PNA. Bis-PNA can bind to specific target sequence to form an internal, triple-stranded invaded complex and a looped-out single strand.

From measurements of linear dichroism, defined as the anisotropic absorption of linearly polarized light (57) , it was early demonstrated that PNA-T₈ can form a right-handed PNA₂/DNA triple helix with poly(dA). The base triplets are arranged in such a fashion that their planes are approximately perpendicular to the helix axis, similar to a standard poly(dA)-(poly(dT))₂ triplex (58) . This PNA₂/DNA triplex structure is remarkably rigid, which indicates an efficient stacking interaction; segments of PNA₂–DNA triplexes, formed at subsaturated stoichiometric strand ratios, are distributed evenly on the poly(dA) strand, separated by flexible single-stranded regions. The formation of (PNA)₂–RNA triple helical structure has been investigated by biological experiments. The T_m values are more or less the same as those of their (PNA)₂–DNA counterparts (59) . It was observed that the translation of CAT-mRNA can be successfully inhibited by the action of a triple helix-forming PNA.

Homopyrimidine PNAs can bind to complementary homopurine target sites in double-stranded DNA by the mechanism of strand invasion (Fig. 2B ). The homopyrimidine PNA recognizes its complementary site in the dsDNA and displaces the pyrimidine strand of dsDNA, forming a stable internal PNA–DNA–PNA triplex involving the homopurine strand and a looped-out, single-stranded structure containing pyrimidine bases. The strand invasion is favored by the presence of internal A-rich regions in a duplex DNA and by moderately low ionic strength (< 50 mM NaCl). Gel retardation assay using DNA restriction fragments containing single PNA targets (1 , 38) showed that strand invasion occurs in a highly sequence-specific manner. The incorporation of a single mismatch in a 10-mer PNA at 20°C reduces the binding constant by more than a factor of 100. The presence of one further mismatch could make the strand invasion virtually impossible.

The formation of a right-handed PNA₂–DNA triplex structure was first concluded from circular and linear dichroism spectroscopic measurements (58) . However, the crystal structure of the PNA₂–DNA triplex reveals considerable deviations from its nucleic acid counterpart (31) . The purine DNA strand forms a Watson-Crick base pair with the homopyrimidine PNA strand in an antiparallel orientation and deploys a second set of base-pairing hydrogen bonds, obeying a Hoogsteen base pairing scheme, to the second PNA strand of identical sequence running parallel with the DNA. There is also another H-bond set between the backbones of those two strands. The Watson-Crick paired PNA–DNA strands in the triplex structure resemble the PNA–DNA duplex. The PNA₂–DNA triplex is wide (diameter ~26 Å) and the pitch is ~16 (triplet) bases. The sugar puckering in the DNA strand is C3'-endo and the backbone torsional angles are similar to those of an A-form structure. The structure is further stabilized through a network of water molecules specifically bound in the minor groove.

Recently, another kind of invasion complex has been reported by Nielsen (60) that requires the presence of nonstandard nucleobases in the PNAs. The complex formed after the binding of PNA molecules is known as double invasion complex, since two PNA strands can simultaneously bind (invade) the two strands (Fig. 2C ).

With the availability of NMR and X-ray crystallography data (20 , 31 , 47 48 49 50) , a few computational studies have been made for further characterization of peptide nucleic acid structures (61 , 62) . The molecular dynamics simulation study of a PNA·DNA-PNA triplex helix in aqueous solution by Shields et al. (62) has been useful by concluding from energetic analysis that the hybrid PNA-DNA-PNA triplex has a characteristic helicity that is quite different from its actual DNA counterpart. The reason is not simply the replacement of sugar phosphate by PNA backbones, but some definite conformational preferences of the PNA strands that have effects on the conformational flexibility. The structure of a PNA·DNA-PNA triple helix is based on a total of 12 acyclic torsion angles, which determine the conformation of adjacent bases in PNA vs. 9 variables, including the sugar ring, in DNA (63) . Moreover, a torsional freedom, absent in the nucleic acid, is also introduced into the structure involving PNA due to the replacement of the sugar ring by a linear bond sequence. The 12 torsional angles are divided into two groups. The first group involves the independent variables, including torsional angles analogous to the glycosyl and ring torsions of the nucleic acids. The second or the dependent set arises in the successful closure of the PNA backbone (63) . The results of these computational (modeling) studies based on predicted structures and cross-verified from the current spectroscopic, calorimetric, and crystallographic data may explain why PNA never forms very B-like structures.

	Antigene and antisense applications of PNA

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Peptide nucleic acids have promise as candidates for gene therapeutic drugs design. They require well-identified targets and a well-characterized mechanism for their cellular delivery. In principle, two general strategies can be adapted to design gene therapeutic drugs (Fig. 5 ). Oligonucleotides or their potential analogs are designed to recognize and hybridize to complementary sequences in a particular gene whereby they should interfere with the transcription of that particular gene (antigene strategy). Alternatively, nucleic acid analogs can be designed to recognize and hybridize to complementary sequences in mRNA and thereby inhibit its translation (antisense strategy). PNAs are chemically and biologically stable molecules and have significant effects on replication, transcription, and translation processes, as revealed from in vitro experiments. Moreover, no sign of any general toxicity of PNA has so far been observed. As we shall see, PNA can interfere with the translation process, and PNA-dsDNA strand displacement complexes can inhibit protein binding and block RNA polymerase elongation.

View larger version (43K): [in this window] [in a new window]   Figure 5. Antigene and antisense strategy. An antigene oligomer (e.g., PNA) could bind to a complementary sequence in the DNA and inhibit transcription of the gene. On the other hand, cells can also be treated with an antisense oligomer, and hybridization to a specific mRNA sequence can inhibit the expression of a protein at the level of translation.

	Inhibition of transcription

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Peptide nucleic acids should be capable of arresting transcriptional processes by virtue of their ability to form a stable triplex structure or a strand-invaded or strand displacement complex with DNA. Such complexes can create a structural hindrance to block the stable function of RNA polymerase and thus are capable of working as antigene agents (Fig. 4) . Evidence from in vitro studies supports the idea that such complexes are indeed capable of affecting the process of transcription involving both prokaryotic and eukaryotic RNA polymerases. PNA targeted against the promoter region can form a stable PNA–DNA complex that restricts the DNA access of the corresponding polymerase. PNA strand displacement complexes, located far downstream from the promoter, can also efficiently block polymerase progression and transcription elongation and thereby produce truncated RNA transcripts; the PNA (DNA poly-purine) target must be present in the gene of interest. Nielsen et al. (64) have demonstrated that even an 8-mer PNA (T₈) is capable of blocking phage T₃ polymerase activity (64) . The presence of a PNA target within the promoter region of IL-2R gene has been used to understand the effect of PNA binding to its target on this gene expression (10 , 65) . The PNA₂/DNA triplex arrests transcription in vitro and is capable of acting as an antigene agent. But one of the major obstacles to applying PNA as an antigene agent is that the strand invasion or the formation of strand displacement complex is rather slow at physiological salt concentrations (36 , 66) . Several modifications of PNA have shown improvement in terms of binding. Modifications of PNA by chemically linking the ends of the Watson-Crick and Hoogsteen PNA strands to each other (28) , introducing pH-independent pseudoisocytosines into the Hoogsteen strand (28) , incorporating intercalators (67) , or positively charged lysine residues (28 , 68) in PNA strand can drastically increase the association rates with dsDNA. Lee et al. (69) have demonstrated that PNA as well as the PNA–DNA chimera complementary to the primary site of the HIV-I genome can completely block priming by tRNA₃Lys. Consequently, in vitro initiation of the reverse transcription by HIV-1 RT is blocked. Thus, oligomeric PNAs targeted to various critical regions of the viral genome are likely to have a strong therapeutic potential for interrupting multiple steps involved in the replication of HIV-1 (69) .

It has been found that under physiological salt conditions, binding of PNA to supercoiled plasmid DNA is faster compared to linear DNA (66 , 68) . This result is relevant to the fact that the transcriptionally active chromosomal DNA usually is negatively supercoiled, which can act as a better target for PNA binding in vivo. It has also been found that the binding of PNA to dsDNA is enhanced when the DNA is being transcribed. This transcription-mediated PNA binding occurs about threefold as efficiently when the PNA target is situated on the nontemplate strand instead of the template strand. As transcription mediates template strand-associated (PNA)₂/DNA complexes, which can arrest further elongation, the action of RNA polymerase results in repression of its own activity, i.e., suicide transcription (70) . These findings are highly relevant for the possible future use of PNA as an antigene agent.

We wish to refer to reports describing the ability of PNA to activate transcription, although this is not actually related to its antigene effect. Mollegaard et al. (71) have efficiently demonstrated that the looped-out single-stranded structure formed as a result of strand invasion is also capable of acting as efficient initiation sites for Escherichia coli and mammalian RNA polymerases in which the polymerase might start transcription using the single-stranded loop as a template (65) . This is consistent with the affinity of RNA polymerase for single-stranded DNA and its ability to transcribe single-stranded DNA.

	Inhibition of translation

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The basic mechanism of the antisense effects by oligodeoxynucleotides is considered to be either a ribonuclease H (RNase H) -mediated cleavage of the RNA strand in oligonucleotide-RNA heteroduplex or a steric blockage in the oligonucleotide–RNA complex of the translation machinery (59) . Oligodeoxynucleotide analogs such as phosphorothioates activate RNase H and thus hold promise of working as antisense agents. However, they also exhibit some nonspecificity in their action. PNA/RNA duplexes, on the other hand, cannot act as substrates for RNase H. Normally, the peptide nucleic acid antisense effect is based on the steric blocking of either RNA processing, transport into cytoplasm, or translation. It has been concluded from the results of in vitro translation experiments involving rabbit reticulocyte lysates that both duplex- (mixed sequence) and triplex-forming (pyrimidine-rich) PNAs are capable of inhibiting translation at targets overlapping the AUG start codon (59) . Triplex-forming PNAs are able to hinder the translation machinery at targets in the coding region of mRNA. However, translation elongation arrest requires a (PNA)₂–RNA triplex and thus needs a homopurine target of 10–15 bases. In contrast, duplex-forming PNAs are incapable of this. Triplex-forming PNAs can inhibit translation at initiation codon targets and ribosome elongation at codon region targets.

Mologni et al. (72) showed effects of three different types of antisense on the in vitro expression of PML/RAR gene. The first one was complementary to the first AUG (initiation) site. The second could bind to a sequence in the coding region that includes the second AUG, the starting site for the synthesis of an active protein. The third PNA was targeted against the 5'-untranslated region (UTR) of the mRNA, the point of assembly of the translation machinery. Together, these three PNAs could efficiently inhibit translation even at a concentration much below the critical concentration used for each individual. The result suggests that the PNA targeting of RNA molecules like PML/RAR requires effective blocking of different sequences on the 5' part of the messenger. A 5'-UTR PNA target can also be used as efficiently as an initiation (AUG) target to achieve an antisense activity of PNA, and a more effective translation inhibition can be achieved by combining PNA directed toward 5'-UTR and AUG regions.

Triple helix-forming PNAs can also hinder the translation process. Bis-PNA or clamp-PNA structures are capable of forming internal triple helical constructs. In principle, if targeted against the coding region of mRNA, PNA₂/RNA triple helix-forming derivatives can also cause a stop in translation, which can be easily verified by the detection of a truncated protein. (59) . However, this methodology requires a sequence optimization for each new target.

Recent studies show that E. coli cells are somewhat permeable for PNA molecules. Good and Nielsen (73 , 74) have shown that it is possible to achieve PNA antisense effects in the ‘leaky’ mutant strains of E. coli. PNAs targeted against the AUG region of the mRNA corresponding to ß-galactosidase and ß-lactamase genes were indeed capable of down-regulating the expression of these two genes (73) . Another study (74) demonstrated the effect of two bis-PNAs, targeted against the homopurine stretches in rRNA, either in the peptidyl transferase center or in the -sarcin loop, in inhibiting the ribosome function in a cell-free system. The translation was arrested at submicromolar range of PNA concentration. The growth of a mutant strain of E. coli, namely, AS19, was also inhibited by using the same PNAs at low micromolar concentration.

	Inhibition of replication

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
It is also possible by using PNA to inhibit the elongation of DNA primers by DNA polymerase. Further, the inhibition of DNA replication should be possible if the DNA duplex is subjected to strand invasion by PNA under physiological conditions or if the DNA is single stranded during the replication process. Efficient inhibition of extrachromosomal mitochondrial DNA, which is largely single-stranded during replication, has been demonstrated by Taylor et al. (75) . The PNA-mediated inhibition of the replication of mutant human mitochondrial DNA is a novel (and also potential) approach toward the treatment of patients suffering from ailments related to the heteroplasmy of mitochondrial DNA. Here wild-type and mutated DNA are both present in the same cell. Experiments have shown that PNA is capable of inhibiting the replication of mutated DNA under physiological conditions without affecting the wild-type DNA in mitochondria.

	Interaction of PNA with enzymes

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
RNase H
The activation of the intracellular enzyme RNase H by oligonucleotides to cleave RNA bound to deoxyribonucleic acid oligomers depends on the chemical structure of RNase H-stimulating oligonucleotides. The antisense oligonucleotide with an RNase H activity (e.g., phosphorothioate oligos) is considered a better antisense molecule (inhibitor) than one without the activity (methylphosphonates and hexitol nucleic acids) (29) . Despite their remarkable nucleic acid binding properties, PNAs generally are not capable of stimulating RNase H activity on duplex formation with RNA. However, recent studies have shown that DNA/PNA chimeras are capable of stimulating RNase H activity. On formation of a chimeric RNA double strand, PNA/DNA can activate the RNA cleavage activity of RNase H (Fig. 6 ). Cleavage occurs at the ribonucleotide parts base-paired to the DNA part of the chimera. Moreover, this cleavage is sequence specific in such a way that certain sequences of DNA/PNA chimeras are preferred over others (29) . They are also reported to be taken up by cells to a similar extent as corresponding oligonucleotides (29) . Thus, PNA/DNA chimeras appear by far the best potential candidates for antisense PNA constructs.

View larger version (14K): [in this window] [in a new window]   Figure 6. Schematic representation of RNase H-mediated cleavage activity after the binding of a PNA–DNA chimera to an RNA target.

Polymerase and reverse transcriptase
In general, there is no direct interaction of PNA with either DNA polymerase or reverse transcriptase. However, different groups have shown indirect involvement of PNA in inhibiting these enzyme functions (activity) under in vitro conditions. For example, PNA oligomers are capable of terminating the elongation of oligonucleotide primers by either binding to the template strand or directly competing with the primer for binding to the template. Primer extension by MMLV reverse transcriptase has been shown to be inhibited by introducing a PNA oligomer (10) . In another experiment, Nielsen et al. (76) demonstrated that the primer extension catalyzed by Taq-polymerase can be terminated by incorporating a PNA oligomer (PNA-H(t)₁₀) into the system. The latter can bind to a (dA)₁₀ sequence in the template and thereby terminate the primer extension. Moreover, uncharged PNA primers with only a single 5'-amino-2',5'-dideoxynucleoside at the carboxyl terminus can be recognized by the Klenow fragment for DNA pol I and VentDNA polymerase (Thermococcus litoralis) (77) , and a linear amplification is possible with the use of an excess of PNA-DNA primer and suitable thermostable polymerases (77) . Moreover, the reverse transcription of gag gene of HIV I is also inhibited in vitro by PNAs (78) . The inhibition has been achieved by using a bis-PNA construct, which is more efficient than the corresponding mono PNA construct (72) . Also, the reverse transcription can be completely inhibited by a pentadecameric antisense PNA, using a molar ratio of 10:1 (PNA/RNA), without any noticeable RNase H cleavage of the RNA (78) .

Telomerase
Human telomerase, a ribonucleoprotein complex consisting of a protein with DNA polymerase activity and an RNA component, synthesizes (TTAGGG)n repeats at the 3' end of DNA strands. PNA oligomers that are complementary to the RNA primer binding site can inhibit the telomerase activity. Studies have shown that the telomerase inhibition activity of PNA is better than that of corresponding activity of phosphorothioate oligonucleotides. This is mainly due to a higher binding affinity of PNA compared to phosphorothioates (79) . Corey and co-workers (80) have demonstrated an efficient inhibition of telomerase after lipid-mediated delivery of template- and nontemplate-directed PNA into the cell.

	PNA as a molecular-biological tool

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Peptide nucleic acids also exhibit potential for use as a tool in biotechnology and molecular biology. Here we will mainly present indications of PNA becoming an important molecular biology tool.

	Enhanced PCR amplification

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The polymerase chain reaction (PCR) has been widely used for various molecular genetic applications including the amplification of variable number of tandem repeat (VNTR) loci for the purpose of genetic typing (81 , 82) . However, in some cases preferential amplification of small allelic products relative to large allelic products presents a problem. This results in an incorrect typing in a heterozygous sample (83) . PNA has been used to achieve an enhanced amplification of VNTR locus D1S80 (6) . Small PNA oligomers are used to block the template, and the latter becomes unavailable for intra- and interstrand interaction during reassociation. On the other hand, the primer extension is not blocked; during this extension, the polymerase displaces the PNA molecules from the template and the primer is extended toward completion of reaction. This approach shows the potential of PNA application for PCR amplification where fragments of different sizes are more accurately and evenly amplified. Since the probability of differential amplification is less, the risk of misclassification is greatly reduced.

Misra et al. (84) demonstrated that PNA–DNA chimera (85) lacking the true phosphate backbone are capable of acting as a primer for the polymerase reaction catalyzed by DNA polymerases. The chimera (PNA)₁₉-T_PG-OH, consisting of a 19 base PNA part linked to a single phosphate-containing dinucleotide (T_PG-OH) with a free 3'-OH terminus, when annealed with a complementary RNA or DNA template strand works as an efficient primer to catalyze the addition of nucleotide by polymerase enzymes. The primer is also recognized by reverse transcriptase and by the Klenow fragment of E. coli DNA polymerase I. The results suggested that the diameter of the duplex region rather than the presence of phosphate backbone of the template primer is the critical factor for a proper template-primer reaction and accommodating the enzyme within the binding domain. It also appears that the primer phosphate backbone may not be essential, at least not in this case, for the polymerase recognition and binding.

	PNA hybridization as alternative to Southern hybridization

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Southern hybridization is perhaps one of the most widely used techniques in molecular biology. Despite its great potential to predict both size and sequence information and information regarding the genetic context, there are certain disadvantages of this process. It requires a laborious multistep washing procedure and there could sometimes be poor sequence discrimination between closely related species. PNA pre-gel hybridization simplifies the process of Southern hybridization by reducing the required time, as the cumbersome post separation, probing, and washing steps are eliminated. Labeled (fluoresceinated) PNA oligomers are used as probes and allowed to hybridize to a denatured DNA sample at low ionic strength. The mixture is thereafter subjected directly to electrophoresis for size separation and single-stranded DNA fragments separated on the basis of length. The charge-neutral PNA allows hybridization at low ionic strength and renders higher mobility to the complex compared to the excess unbound PNA. The DNA-PNA hybrids are blotted (transferred) onto a nylon membrane, dried, UV cross-linked, and detected using standard chemiluminescent techniques (8) . Alternatively, the bound PNA can be detected by using capillary electrophoresis (vide infra), which can make use of the direct fluorescence detection method. Under the same conditions, a normal DNA–DNA duplex will tend to disrupt whereas the PNA–DNA duplex will remain intact due to the strong binding of PNA to DNA. This allows specific sequence detection with simultaneous size separation of the target DNA following a simple and straightforward protocol. Consequently, the analysis is much faster than conventional the Southern hybridization technique.

	PNA-assisted rare cleavage

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Peptide nucleic acids, in combination with methylases and other restriction endonucleases, can act as rare genome cutters (9) . The method is called PNA-assisted rare cleavage (PARC) technique (Fig. 7 ). It uses the strong sequence-selective binding of PNAs, preferably bis-PNAs, to short homopyrimidine sites on large DNA molecules, e.g., yeast or DNA. The PNA target site is experimentally designed to overlap with the methylation/restriction enzyme site on the DNA, so a bound PNA molecule will efficiently shield the host site from enzymatic methylation whereas the other, unprotected methylation/restriction sites will be methylated. After the removal of bis-PNA, followed by restriction digestions, it is possible to cleave the whole DNA by enzymes into limited number of pieces (9) . DNA is efficiently protected from enzymatic digestion due to methylation in most of the sites except for those previously bound to PNA. Thus, short PNA sequences, particularly positively charged bis-PNAs, in combination with various methylation/restriction enzyme pairs can constitute an extraordinary new class of genome rare cutters.

View larger version (43K): [in this window] [in a new window]   Figure 7. The PNA-assisted rare cleavage technique: Symbols (red straight horizontal line), (filled red square), (filled gray square) represent PNA binding site, restriction enzyme site, and methylated restriction enzyme site, respectively. Methylated sites are not subjected to a restriction digestion.

	Artificial restriction enzyme system

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
S1 nuclease cleaves single-stranded nucleic acids releasing 5'-phosphoryl mono- or oligonucleotides. It removes the single-stranded overhangs of DNA fragments and can be used in RNA transcript mapping and construction of unidirectional deletions. PNA in combination with S1 nuclease can work as an ‘artificial restriction enzyme’ system. Homopyrimidine PNA oligomers hybridize to the complementary targets on dsDNA via a strand invasion mechanism, leading to the formation of looped-out noncomplementary DNA strands. The enzyme nuclease S1 can degrade this single-stranded DNA part into well-defined fragments. If two PNAs are used for this purpose and allowed to bind to two adjacent targets on either the same or opposite DNA strands, it will essentially open up the entire region, making the substrate accessible for the nuclease digestion and thereby increasing the cleavage efficiency (5) .

	Determination of telomere size

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The conventional method for the determination of telomere length involves Southern blot analysis of genomic DNA and provides a range for the telomere length of all chromosomes present. The modern approach uses fluorescein-labeled oligonucleotides and monitor in situ hybridization to telomeric repeats. However, a more delicate approach resulting in better quantitative results is possible by using fluorescein-labeled PNAs, as shown by Lansdorp et al. (86) . This PNA-mediated approach permits accurate estimates of telomeric length. In situ hybridization of fluorescein-labeled PNA probes to telomeres is faster and requires a lower concentration of the probe compared to its DNA counterpart. Low photobleaching and an excellent signal-to-noise ratio make it possible to quantitate telomeric repeats on individual chromosomes in this way. Experiments suggest that variations of this approach can possibly be applied to other repetitive sequences.

	Nucleic acid purification

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Based on its unique hybridization properties, PNAs can also be used to purify target nucleic acids. PNAs carrying six histidine residues have been used to purify target nucleic acids using nickel affinity chromatography (7) . Also, biotinylated PNAs in combination with streptavidin-coated magnetic beads may be used to purify Chlamydia trachomatis genomic DNA directly from urine samples. However, it appears that this simple, fast, and straightforward ‘purification by hybridization’ approach has certain drawbacks. It requires the knowledge of a target sequence and depends on a capture oligomer to be synthesized for each different target nucleic acid. Such target sequences for the short pyrimidine PNA, i.e., the most efficient probe for strand invasion, are prevalent in large nucleic acids. Thus, short PNAs can also be used as generic capture probes for purification of large nucleic acids. It has been shown that a biotin-tagged PNA-thymine heptamer could be used to efficiently purify human genomic DNA from whole blood by a simple and rapid procedure.

	PNA as a diagnostic tool

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The high-affinity binding of PNA oligomers has led to the development of new applications of PNA, especially as a diagnostic probe for detecting genetic mutations: applications are possible for the detection of genetic mutation and mismatch analysis that can use its unique hybridization properties. The ensuing sections will highlight some of the recent developments related to the use of PNA as a probe to detect genetic mutations and corresponding mismatch analysis confirming its potential as a diagnostic tool for clinical applications.

	Single base pair mutation analysis using PNA-directed PCR clamping

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Amplification of the target nucleic acid by the PCR technique is considered an important step for detection of genetic diseases. The higher specificity of PNA binding to DNA, higher stability of a PNA–DNA duplex compared to the corresponding DNA–DNA duplex, and its inefficiency to act as a primer for DNA polymerases are the bases for this novel technique. This strategy (Fig. 8 ) includes a distinct annealing step involving the PNA targeted against one of the PCR primer sites. This step is carried out at a higher temperature than that for conventional PCR primer annealing where the PNA is selectively bound to the DNA molecule. The PNA/DNA complex formed at one of the primer sites effectively blocks the formation of a PCR product. PNA is also able to discriminate between fully complementary and single mismatch targets in a mixed target PCR. Sequence-selective blockage by PNA allows suppression of target sequences that differ by only one base pair. Also, this PNA clamping was able to discriminate three different point mutations at a single position, as demonstrated in a model system by Örum et al. (13) .

View larger version (15K): [in this window] [in a new window]   Figure 8. Mutation analysis using PNA-directed PCR clamping: schematic representation of the strategy for the PCR cycle involving PNA-directed clamping. In the case of the normal (wild-type) DNA, the bound PNA will sterically hinder annealing of a partially overlapping primer sequence, thus preventing the normal sequence from appropriate PCR amplification. In the case of mutant alleles, the melting temperature of the PNA/DNA is reduced and the primer can out-compete PNA annealing to carry on preferential amplification of mutant sequences.

Thiede et al. (15) have reported a novel approach for simple and sensitive detection of mutations in the ras proto-oncogenes. Their strategy, which is also based on the principle described by Örum et al. (13) , is schematically presented in Fig. 8 . Chromosomal DNA is hybridized to a 15-mer PNA probe complementary to a normal wild-type sequence surrounding codons 12 and 13. For the wild-type K_i-ras, formation of PNA/DNA duplex will be favored, which in turn would sterically hinder the annealing of overlapping oligonucleotides preventing normal K_i-ras sequences from being sufficiently (PCR) amplified. On the other hand, in the case of the mutant alleles, the melting temperature of the mutated or mismatched PNA-DNA hybrid is much lower than the corresponding normal one. Hence, the binding of primer oligonucleotide will be favored to out-compete PNA annealing. Consequently, mutated sequences will be preferentially amplified (15) .

One major advantage of this PNA-mediated PCR clamping is that it allows detection of mutations stretched over 4–6 bp regions in a single reaction, and this could also be used to detect other hot-spot mutations (13 , 15) .

	Screening for genetic mutations by capillary electrophoresis

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
In capillary electrophoresis (87 88 89) , the separation is generally carried out using a long, thin fused silica capillary (typically 50–80 cm long, inner diameter ~ 10–300 µm). A portion of the coating, close to one end of the capillary, is removed to allow optical detection of the analyte. The analyte passes the detection window during a separation process and can be visualized by online, automated, UV, or laser-induced fluorescence (LIF) detection systems. Capillary electrophoresis is capable of analyzing minute amounts of sample (typically on the order of picograms to femtograms). However, it is not possible to analyze more than one sample at a time (for details, see refs 87 88 89 ), which is regarded as the major disadvantage compared to slab gel electrophoresis.

A novel diagnostic method for the detection of genetic mutation using PNA as a probe for capillary electrophoresis has been reported by Carlsson et al. (14) . The method is sensitive enough to detect a single mismatch in the sample DNA. The model system consisted of four 50-mer, single-stranded DNA fragments representing a part of the cystic fibrosis transmembrane conductance regulator gene, one wild-type and three mutant sequences (Table 2 ), and a 15-mer PNA probe having a sequence complementary to the wild-type oligomer. The probe PNA only binds to the fully matched DNA, and the presence of this duplex is detected using free solution capillary electrophoresis (14) . Separation of full match from mismatch duplexes was accomplished at a high temperature (~70°C; Fig. 9 ) and 50 mM ionic strength. At this temperature and ionic strength, only the hybrid duplex carrying the wild-type DNA sequences remains stable, while the hybrid complex carrying single mismatch DNA will be melted. Free PNA binds to the capillary wall and is not detected. In another related experiment, the same PNA probe was added to a PCR preparation of the 142-mer wild-type CF fragments and the 139-mer three-base deletion fragments. At 50°C, the wild-type PNA–DNA duplex was identified whereas no signal at all was seen for the mutant. Nordén and co-workers (unpublished observations) have also used free solution capillary electrophoresis to detect point mutation in a 15-mer DNA fragment representing a vulnerable part of the p53 gene. The 15-mer PNA probe was complementary to the wild-type p53 sequence (in this case, the 15-mer oligonucleotide). The experimental temperature was lower than the thermal melting temperature of the fully matched PNA-DNA hybrid, but higher than the melting temperature of the PNA–DNA duplex with a mismatch at the 8th position. (The mismatch corresponds to a mutation in the codon 175 of the p53, resulting in an altered P53 protein with an arginine replaced by a histidine.) At this temperature, the fully matched duplex maintained its structural integrity but the PNA probe could not bind to the single mismatch DNA sequence, so the fully matched duplex could be separated from the mismatch complexes. This method is able to detect single-base substitutions. A big advantage of using PNA instead of DNA as a hybridization probe is that it provides a more efficient way of sequence discrimination and allows separation at high temperature at which DNA-DNA hairpin structures, responsible for band compression in gel electrophoresis, are eliminated.

View this table: [in this window] [in a new window]   Table 2. Synthetic DNA fragments of the cystic fibrosis gene and corresponding PNA probe (14)

View larger version (19K): [in this window] [in a new window]   Figure 9. Free solution capillary electrophoresis of a mixture of PNA and DNA. A) The electrophoregram at 70°C of a mixture of PNA and DNA (MU1, Table 2 ). The latter has a single-base substitution in the recognition region. At 70°C, PNA cannot remain bound to the mutant DNA sequence and the peak corresponding to PNA–DNA hybrid duplex will not appear. B) Control electropherogram at the same temperature representing the interaction of the same PNA (Table 2) with the wild-type DNA sequence. Adapted by permission from Carlsson et al., 1996 (ref 14 ).

PNA probes with fluorescent tags offer sensitive detection and require only a very low concentration of the sample. At high temperature (as described above), the LIF detection system will generate a signal for the bound PNAs since free PNA is efficiently removed. It might be possible in the future to establish a universal screening strategy for any genetic disease with a known spectrum of mutations by developing a PNA probe library, possibly using multiple fluorescent tags for multiplex testing of one or more exons.

The RNA binding properties of PNAs have also been characterized using capillary gel electrophoresis (90) . Like free solution capillary electrophoresis, only a minute amount (~ fmol) is required for analysis. Compared to free solution capillary electrophoresis, this method shows a greater dependence on mass. This method, which can quantitatively resolve free and bound PNA, enables the measurement of relative binding kinetics using gel-filled capillaries.

Another gel-based strategy, commonly known as affinity electrophoresis, can be exploited in PNA-based diagnostics. Depending on the noncovalent interaction of two species, this requires the immobilization of one of the interacting species in a gel matrix (91) . If PNA is entrapped in the gel matrix, it can cause a retardation of the complementary oligonucleotides and allow specific hybridization during electrophoretic passage of a complementary DNA. The retardation of DNA is directly related to the binding of PNA through complementarity. Hence, the PNA-DNA hybridization in the gel matrix is also sensitive to mismatch. The method allows the visualization of hybridization between an immobilized PNA and a complementary DNA, migrating in an electric field, in real time. Moreover, a more sensitive detection of a reasonable number of samples in real time is possible by using the LIF detection system using fluorescent samples.

	PNA as a probe for nucleic acid biosensor

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
The DNA biosensor technology holds promise for rapid and cost-effective detection of specific DNA sequences. A single-stranded nucleic acid probe is immobilized onto optical, electrochemical, or mass-sensitive transducers to detect the complementary (or mismatch) strand in a sample solution. The response from the hybridization event is converted into a useful electrical signal by the transducer. We describe here the use of PNA as a novel probe for sequence-specific biosensors and highlight some of the promise it holds to work as the recognition layer in DNA biosensors.

BIAcore technique
The PNA hybridization and corresponding mismatch analysis can be studied using a BIAcore (biomolecular interaction analysis) instrument (92) , which can evaluate a real-time biomolecular interaction analysis using optical detection technology. The real-time interactions are monitored on a sensor (surface) chip, which constitutes the core part of a BIAcore instrument. The probe molecule is attached directly to the surface and the analyte molecule is free in solution. The detection principle in BIA uses surface plasmon resonance (92) . The response signal of the BIAcore apparatus is proportional to the change in the refractive index at the surface and is assumed to be proportional to the mass of substance bound to the chip.

The first report regarding the study of PNA-DNA/RNA hybridization using the BIAcore technique came from the work of Jensen et al. (42) in 1997. The sensor chip used in this case was basically a thin gold surface covered with a layer of dextran and containing streptavidin chemically coupled to the dextran (Pharmacia sensor chip SA5). A biotinylated PNA (biotin-(eg1)₃-TGTACGTCACAACTA-NH₂) probe was immobilized on the surface by using the strong coupling between biotin and streptavidin. The amount of bound substance (fully complementary as well as various singly mismatched RNA and DNA oligonucleotides) was measured as a function of time when a solution containing the complementary strands passed over the chip surface. In this way the association kinetics could be studied. The dissociation was subsequently studied by washing the surface with appropriate buffer and monitoring the time dependence of the mass decrease. Assuming a two-state model, A + B AB, analysis of the hybridization kinetics was carried out. The PNA surface can be regenerated in SA5 by removing the remaining hybridized products with HCl. Thus, consecutive studies could be carried out with the same immobilized PNA.

Nordén and co-workers (unpublished observations) have also explored the possibility of carrying out BIAcore measurements using a plain gold surface (e.g., BIAcore sensor chip J1) to which PNA molecules carrying cystein at the amino-terminal can be immobilized using the strong coupling between gold and sulfur. In this way, erroneous results due to nonspecific binding of ligands to the dextran layer, if any, can be eliminated. However, it should be kept in mind that DNA has a high affinity for gold and generally is nonspecifically adsorbed to the surface. Direct addition of analyte DNA molecules onto the sensor surface to study its binding with PNA might facilitate its adsorption to the gold surface. Short spacer molecules, e.g., mercaptohexanol, can be used together with the ligand (probe) to form the PNA monolayer at the top of the sensor (gold) surface to prevent DNA from being nonspecifically adsorbed to the surface.

Quartz crystal microbalance (QCM)
The quartz crystal microbalance has been used for some time to monitor mass or thickness of thin films deposited on surfaces, study gas adsorption and deposition on surfaces in the monolayer and submonolayer regimes (93) , research areas related to electrochemistry, or study protein adsorption. Only recently has this sensitive mass measuring device begun to be used in the area of biochemistry and biotechnology, such as for studying the hybridization of nucleic acids on surface (94 95 96) . The resonant frequency of the crystal changes due to a minute weight increase on the surface. It is expected that immobilized PNA strands (or probes) would show an improved distinction between the closely related target sequences compared to an immobilized DNA probe. A recent report by Wang and co-workers (97) on quartz crystal microbalance biosensor, based on peptide nucleic acid probes, showed that the system can differentiate between a full complementary and single mismatch oligonucleotide. A rapid and sensitive detection of mismatch sequences is possible by monitoring the frequency/time response of the PNA-QCM biosensor. The PNA probes used in the above-mentioned study (97) , which formed the monolayer onto the gold QCM surface, contained a cysteine attached to the PNA core with the help of an ethylene glycol unit. The remarkable specificity of the immobilized probe provides a rapid hybridization with corresponding oligonucleotides. Such a mismatch sensitivity of PNA-immobilized QCM biosensors could be of great importance for diagnostic applications, particularly for genetic screening and diagnosis of malignant diseases.

MALDI-TOF mass spectrometry
MALDI-TOF mass spectrometry (for nucleic acid-based applications, see refs 32 , 98 , 99 ) has been used successfully in PNA-based diagnostic research to study discrimination of single-nucleotide polymorphisms (SNPs) in human DNA (33) . Human genomic and mitochondrial DNA contain many SNPs that may be linked to diseases. Rapid and accurate screening of important SNPs, based on high-affinity binding of PNA probes to DNA, is possible by using MALDI-TOF mass spectroscopy. The captured, single-stranded DNA molecules are PCR-amplified and thereafter hybridized with PNA probes in an allele-specific fashion. MALDI-TOF can rapidly and accurately detect (identify) these hybridized PNA probes. This provides a straightforward, rapid, accurate, and specific detection of SNPs in amplified DNA (33 , 100) . The detection of multiple point mutations using allele-specific, mass-labeled PNA hybridization probes is also possible by using a direct MALDI-TOF-MS analysis method (101) . The mass spectra will show peaks of distinct masses corresponding to each allele present, and in this way a mass spectral ‘fingerprint’ of each DNA sample can be obtained.

Potentiometric measurements
Wang et al. (16) have also reported the use of PNA as a recognition probe for the electrochemical detection of the hybridization event using chronopotentiometric measurements. The method consists of four steps: probe (PNA) immobilization onto the transducer surface, hybridization, indicator binding, and chronopotentiometric transduction. A carbon paste electrode is in this process containing the immobilized DNA or PNA probe. The hybridization experiment was carried out by immersing the electrode into the stirred buffer solution containing a desired target, followed by measurement of signal.

	Cellular effects and delivery of PNA

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
It is important to understand the effect of peptide nucleic acids on intact cells and problems related to its delivery into the cell. The cellular uptake of this unique nucleic acid analog is very slow, which is still considered to be the major challenge that must be overcome before it can be used as a therapeutic drug. So far, there is hardly any report of the antisense activity of PNA in cell culture without the use of brute techniques to help bypass the membrane barrier.

Effects of PNAs on intact cells have been demonstrated on cellular microinjection; antisense activity against a transfected gene has also been established in this way (10 , 18) . Serious efforts are being made to increase the cellular uptake of PNA, particularly by modifying the molecule itself or conjugating to it suitable potential ligand molecules that could enhance a physical or receptor-mediated cellular uptake (102 103 104) . Incorporation of a ‘guide’ sequence or some ‘vector’ peptides is one potential approach whereby PNA is attracted to the cell membrane and helped in docking into it. Several methods have been proposed to facilitate the uptake of PNAs in eukaryotic cells. These include transient permeabilization with streptolysin O (102) , cell membrane permeabilization by lysolectin (12) or detergents like Tween (79) , or conjugation with peptides capable of being internalized easily (103 104 105) .

Recently, Aldrian-Herrada et al. (106) showed that peptide nucleic acids are rapidly internalized in culture neurons when coupled to a delivery peptide [retro-inverso (107 , 108) analog of the 16-mer p-Antp peptide]. A PNA antisense for the AUG translation initiation region of prepro-oxytocin mRNA was coupled to the vector retro-inverso peptide. The conjugated construct was internalized by cultured cerebral cortex neurons within minutes whereas the penetration of nonconjugated single PNA molecules was rather slow. Both PNA and the peptide-PNA construct lowered the amounts of mRNA coding for prepro-oxytocin in these neurons. This result is promising and demonstrates that PNAs guided by suitable vector peptides could work as antisense agents. Corey and co-workers (80) have reported a novel method for in vitro cellular delivery of peptide nucleic acids using a cationic lipid. The cationic lipid is capable of associating with the negatively charged phosphodiester backbone of DNA and RNA and fusing with the cell membrane to allow the oligonucleotide to enter into the cell through an endocytotic pathway. This technique has been improvised for the delivery of PNA molecules into the cells. Desired PNA oligomers are hybridized to overlapping oligonucleotides and the complex is mixed with cationic lipid (80) . The cationic lipid–DNA–PNA complex thus formed can be internalized, and the partially hybridized PNA is imported into the cell as a passive cargo (80) . On passive delivery into the cell, peptide nucleic acid is expected to dissociate itself from the complex.

Another strategy that has been adapted to improvise the delivery of PNA in vitro is to incorporate it into delivery vehicles (vesicles), e.g., liposomes.

There are also some reports of direct PNA uptake in rat embryo fibroblasts (109) and human myoblasts (75) , although at extremely high exposure concentrations (~20 µM). The poor cellular uptake of naked regular PNA is still considered a major obstacle against the prospective use of PNA as a gene therapeutic drug. Peptide nucleic acid–nucleic acid chimeras are reported to be taken up by Vero or NIH3T3 cells even at a relatively lower extracellular concentration (1 µM), which leaves us with the idea that a PNA–DNA chimera may be a better antisense agent. The kinetics of uptake is similar to that observed for an oligonucleotide of the same sequence. At a higher concentration of PNA, cytotoxic effects could also be observed (110 , 111) .

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 
Peptide nucleic acids provide a novel class of compounds with wide biological potential. Their very specific interactions with DNA and RNA and their chemical and biological stability make them promising both as therapeutic lead compounds and agents for diagnostic applications. The application of PNA as a genetic therapeutic agent has to await the development of efficient and safe methods for its uptake and cell penetration. By contrast, its application as a recognition molecule has already led to promising developments in many areas of chemistry, biology, and biotechnology (74 , 75) . Recent efforts to provide further applications of this exciting nucleic acid analog include modifications of backbone and the development of novel base analogs (29 , 85 , 112 113 114 115 116 117 118 119 120) .

	REFERENCES

TOP ABSTRACT INTRODUCTION Chemical properties and related... Synthesis and purification Physicochemical properties PNA-DNA/RNA and PNA-PNA duplexes (PNA)2-DNA triplexes Antigene and antisense... Inhibition of transcription Inhibition of translation Inhibition of replication Interaction of PNA with... PNA as a molecular-biological... Enhanced PCR amplification PNA hybridization as alternative... PNA-assisted rare cleavage Artificial restriction enzyme... Determination of telomere size Nucleic acid purification PNA as a diagnostic... Single base pair mutation... Screening for genetic mutations... PNA as a probe... Cellular effects and delivery... CONCLUSIONS REFERENCES

 

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