A general overview of features and applications of PNAs

Peptide Nucleic Acids (PNAs) are synthetic mimics of DNA in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. These molecules bind to complementary DNA and RNA sequences with high affinity and specificity, forming complexes with extraordinary chemical and biological stability. First described in 1991, PNAs have received great attention due to their several favorable properties. The unique physico-chemical and biological characteristics of PNA, open up many important biological, diagnostic and therapeutics applications, not achievable with traditional oligonucleotides. INTRODUCTION Peptide Nucleic Acids (PNAs) are analogs of DNA in which the negatively charged sugar-phosphate backbone is replaced by a neutral “peptide-like” skeleton consisting of repeated N-(2-aminoethyl)-glycine units linked by peptide bonds, resulting in an achiral and uncharged mimic. The four natural nucleobases are connected to the central ammine of the backbone by methylene carbonyl linkages and come off the backbone at equal spacing to the DNA bases. PNAs were first described by Nielsen’s group in 19911. These molecules were designed using computer modelling, in order to generate agents able to recognize double-stranded DNA sequences via triple helix formation, with the intent of control gene expression in connection with the development of antigene therapeutic drugs2. The outcome of this study led to a new type of DNA mimetic that besides the ability to form triple helixes can also interact with complementary single-stranded RNA and DNA with high affinity and specificity, obeying the Watson-Crick hydrogen bonding rules3. These biophysical properties, together with high chemical and enzymatic stability and low toxicity4,5 make PNA one of the most valuable nucleic acid mimetics, with a high potential for application both as a genetic diagnostic and as a therapeutic in the anti-sense and anti-gene strategy4,5. Conversely, the applicability of PNA is hampered by low water-solubility, a tendency to form aggregates and, in particular, poor cell membrane permeability6. To overcome these drawbacks, much attention has been focused on the development of PNA conjugates. PNAs have been conjugated to a variety of effector molecules. Among these, small peptide fragments, often derived from functional proteins, are able to convey their specific properties to the conjugate3. BIOPHYSICAL CHARACTERISTICS Binding properties PNAs bind to complementary nucleic acids sequences following Watson-Crick base pairing with the PNA and DNA strands joined through hydrogen bonds. Owing to their neutral backbone and proper interbase spacing, PNAs bind to their complementary nucleic acid sequence with higher affinity and specificity compared to traditional oligonucleotides2. The lack of electrostatic repulsion between PNA and DNA strands makes binding between PNA and DNA or RNA much more stronger than that between DNA and DNA or RNA7. The greater affinity of PNA/DNA or PNA/RNA complexes is reflected in their higher thermal stability. Experiments done with PNA sequences showed that the melting temperatures (Tm) are higher for PNA hybrids than for either DNA/DNA or DNA/RNA7. On average, the Tm of a PNA/DNA duplex is 1°C higher for base pair compared to that of the corresponding DNA/DNA or DNA/RNA duplex7. Generally a 10-mer PNA has a Tm of about 50°C and a 15-mer of about 70°C2. The thermal stability of a PNA/DNA duplex is essentially independent of the salt concentration in the hybridization solution8. Unlike DNA, PNA does not need proper salt concentration for hybridization. The Tm of a 15-mer duplex decreases by only 5°C as the NaCl concentration is raised from 10 mM to 1M9. Such independence of the ionic strength is a result of the uncharged nature of the PNA backbone. PNA can act as a good probe to detect target sequence of secondary structure which is not stable in low salt concentration. Due to the higher affinity for complementary nucleic acids sequences, shorter PNAs can be used as probes. A typical PNA probe is 13-17-mer instead of 20-25-mer wich are the typical length of DNA probes9. Furthermore the stronger binding properties of PNA should decrease the amount of antisense needed for the inhibition of gene expression. In addition to their higher binding properties, PNAs also show greater specificity in binding to complementary DNAs. The thermal stability of the PNA/DNA duplexes is strongly affected by the presence of imperfect matches2. The presence of mismatches in PNA/DNA duplex is much more destabilizing than a mismatch in a DNA/DNA duplex. For example, a single base mismatch results in 15°C and 11°C lowering of the Tm of a 15mer PNA/DNA and DNA/DNA duplex, respectively2. This property of PNA is responsible for the remarkable discrimination between perfect matches and mismatches offered by PNA probes, and makes them so attractive as oligonucleotide recognition elements in biosensor technology2. In conclusion, the greater stability of PNA/DNA or PNA/RNA complexes is reflected in the higher Tm over the corresponding DNA/DNA or DNA/RNA duplexes, while the greater specificity is reflected in the larger decreases in Tm caused by a single mismatch in the base sequences. In addition PNA/DNA or PNA/RNA hybridization reaction is over 100 times faster than DNA/DNA or DNA/RNA hybridization (hybridization time, PNA: 30-45 minute while DNA: 3 hours to overnight)9. High affinity of binding and high rates of association combine to give PNAs a remarkable propensity for invasion of duplex DNA, forming triple-stranded structures10. The formation of triple-stranded complex by invasion is comprised of a PNA/DNA double helix (formed by Watson-Crick hydrogen bonds) with a second PNA strand lying in the major groove of the duplex (formed by Hoogsten hydrogen bonds). The stability of these triple helix structures is so high that 2 PNA molecules can displace a duplex DNA strand to form a triplex with the complementary fragment10. In general, a 10-mer homopyrimidine PNA binds to its target with Tm upper than 70°C4. The conditions that favor triple helix formation are: many pyrimidines and low pH (4.5 to 6.5)10. However a triple helix can also be formed even if there is a couple of purines in the sequence4. PNA-DNA/RNA and PNA-PNA duplexes The thermal melting temperature (Tm), defined as the temperature at which 50% of the complexes have been dissociated9, gives an idea of the stability of the PNA-DNA or PNA–RNA duplex. The thermal stability of a PNA–RNA duplex is even higher than that of a PNA–DNA duplex. 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 increases9. 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 hetero-duplex is considerably lowered by the presence of mismatches9. 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. 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 usually lies between that of the corresponding PNA–DNA and DNA–DNA duplexes9. 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. 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 possible9. Biological and chemical stability PNAs are resistant to enzymatic degradation11. Their peptide backbone bearing purine and pyrimidine infact is not easily recognized by nucleases and proteases. Since they are not easily degraded by enzymes, the lifetime of these polymers is extended both in vitro and in vivo11. In addition to biological stability, PNAs are also very stable against various chemicals and over a wide range of pH. Unlike DNA wich is susceptible to depurination at acidic pH (pH 4.5 ~ 6.5) PNAs are fairly acid stable11. PNAs are also stable in a wide range of temperature11. PNA 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 oligomer7. SYNTHESIS AND PURIFICATION 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 using, for example, a (methylbenzhydryl) amine polystyrene resin as the solid support12,13,14,15,16. The scheme for protecting the amino groups of PNA monomers is based on either Boc or Fmoc chemistry. The postsynthetic modification of PNA uses coupling of a desired group to an introduced lysine or cysteine residue in the PNA. 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 subsequent generation of PNAs might contain an unusual modification of the backbone or a chimeric Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 23 nr 3 • Focus on Peptides & Amino Acids 15 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 structures9. 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) purification12,17,18,19. The crude PNA products can also be cleaved from the solid matrix by treatment with trifluoroacetic acid and mcresol (4:1) mixture12,18,19. Then PNA is precipitated by ice-cold diethyl ether and dissolved in 0.1% trifluoroacetic acid solution. Further purification and subsequent characterization are done using reverse-phase HPLC, followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry12,17,18,19. Some recent modifications, including the incorporation of positively charged lysine residues (carboxylterminal or backbone modification in place of glycine), have shown improvement as to solubility9. Negative charges may also be introduced, especially for PNA–DNA chimeras, which will enhance the water solubility9. The addition of peptide sequences to PNAs is a convenient method for obtaining chimeric molecules in which the peptide domain enhances hybridization or cell uptake of the attached PNA13,20. Peptides can be added to a PNA in a number of ways. If the peptide contains only three or fewer different amino acids, it can be conveniently added immediately before or after synthesis21. If more than three different amino acids need to be added, it is often more convenient to contract a dedicated peptide synthesis facility to add the completed peptide21. The thiol group of cysteine provides a convenient reactive group for PNA modification21. PNA–peptide conjugates can be assembled by adaptation of established solid-phase peptide synthesis protocols3. DELIVERY PNAs do not cross phospholipid bilayers spontaneously21,22. Instead, delivery to cells requires modification to the PNA to allow co-administration with cationic lipids, or conjugation to cell-permeant peptides21,23. Cellular delivery of unmodified PNAs Microinjection provides an excellent tool for assessing the validity of PNA as an antisense agent. It is a laborious technique, which is only applicable for small-scale experimental set-ups6. A more feasible method for transfer of PNA to cells is offered by electroporation, which has been used successfully in several studies6. Co-trasfection with DNA is another way of transfecting unmodified PNAs to cells6. Even permeabilization of eukaryotic cells by streptolycin-O can make these permeable for unmodified PNA6. Unmodified PNAs have also been successfully delivered directly to cells without the use of transfection protocols6. In eukaryotes, simple addition to the culture medium can also in some cases result in a significant cellular uptake of unmodified PNAs6. The basis for the cellular uptake of PNA in this system is yet not understood. However, the extremely high concentrations of PNA may promote a subtoxic stress inducing less than normal stringency in processes such as endoand pinocytosis6. Cellular delivery of modified PNAs As just described, a number of protocols now exist for cellular delivery of unmodified PNA6. However, faster progress in the development of PNA as an antisense agent depends on finding more efficient and more general delivery protocols. Furthermore, development of PNA-based drugs most likely requires that cell permeability can be conferred by the PNA molecule itself. Several modifications of PNA have been introduced in an attempt to meet these demands6,7,24,25. Some examples can be conjugation to lipophilic moieties23,25, to peptides, to cell-specific receptor ligands6. In future therapeutical applications it might be of advantage if PNA can be delivered to specific (eukaryotic) cells. In this way the risk of adverse side-effects in non-targeted cells would be avoided. APPLICATIONS PNAs are a powerful molecular tool with a wide range of important applications. Due to their ability of interacting with high sequence specificity to a chosen target in a gene sequence, they are of major interest in medicinal and biotechnological contexts7,9,26-30. They show promise for the development of gene therapeutic agents9,31,32, diagnostic devices for genetic analysis9, and as molecular tools for nucleic acid manipulations9,32,33-37. In vitro studies indicate that PNAs could inhibit both transcription and translation of genes to which they have been targeted, which holds promise for their use for antigene and antisense therapy7,9,28,31,32. Antigene and antisense applications Peptide nucleic acids have promise as candidates for gene therapeutic drugs design28,31,32. PNAs can be designed to recognize and hybridize to complementary sequences in a particular gene, interfering with its transcription (antigene strategy)7,9,28, or can be designed to recognize and hybridize to complementary sequences in mRNA, inhibiting its translation (antisense strategy)7,9,28. Inhibition of transcription: 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. Such complexes are indeed capable of Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 23 nr 3 • Focus on Peptides & Amino Acids 16 affecting the process of transcription involving both prokaryotic and eukaryotic RNA polymerases7,9,28. Inhibition of translation: Normally, the peptide nucleic acid antisense effect is based on the steric blocking of either RNA processing, transport into cytoplasm, or translation7,9,28. Inhibition of replication: It is also possible by using PNA to inhibit the elongation of DNA primers by DNA polymerase7,9. Furthermore, 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 process9. Interaction of PNA with enzymes: Despite their remarkable nucleic acid binding properties, PNAs generally are not capable of stimulating RNase H activity on duplex formation with RNA38. However, recent studies have shown that DNA/PNA chimeras are capable of stimulating RNase H activity38. 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 under in vitro conditions39,40. 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 activity41. Studies have shown that the telomerase inhibition activity of PNA is better than that of corresponding activity of phosphorothioate oligonucleotides41. This is mainly due to a higher binding affinity of PNA compared to phosphorothioates9,41. PNA as a molecular-biological tool Peptide nucleic acids also exhibit potential for use as a tool in biotechnology and molecular biology. Enhanced PCR amplification: The polymerase chain reaction (PCR) has been widely used for various molecular genetic applications42-46. Small PNA oligomers are used to block the template, so the latter becomes unavailable for intraand interstrand interaction during reassociation9. 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. PNA–DNA chimera, lacking the true phosphate backbone are capable of acting as a primer for the polymerase reaction catalyzed by DNA polymerases47,48. The primer is also recognized by reverse transcriptase and by the Klenow fragment of E. coli DNA polymerase I9. Probabily 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 for the polymerase recognition and binding9. PNA hybridization as alternative to Southern hybridization: Southern hybridization is perhaps one of the most widely used techniques in molecular biology. Despite its great potential to predict both size and sequence, and information regarding the genetic context, there are certain disadvantages in this process9. 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 eliminated27,49. PNA-assisted rare cleavage: Peptide nucleic acids, in combination with methylases and other restriction endonucleases, can act as rare genome cutters43. The method is called PNA-assisted rare cleavage (PARC)43. It uses the strong sequence-selective binding of PNAs, preferably bis-PNAs, to short homopyrimidine sites on large DNA molecules. Artificial restriction enzyme system: S1 nuclease cleaves single-stranded nucleic acids releasing 5’-phosphoryl monoor oligonucleotides51. 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 efficiency51. Determination of telomere size: 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 Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 23 nr 3 • Focus on Peptides & Amino Acids 17 present9. 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 PNAs26,27,52. Nucleic acid purification: Based on its unique hybridization properties, PNAs can also be used to purify target nucleic acids9. PNAs carrying six histidine residues have been used to purify target nucleic acids using nickel affinity chromatography53. Also, biotinylated PNAs in combination with streptavidin-coated magnetic beads may be used to purify microbial genomic DNA. However, it appears that this simple, fast, and straightforward ‘purification by hybridization’ approach has certain drawbacks9. It requires the knowledge of a target sequence and depends on a capture oligomer to be synthesized for each different target nucleic acid. PNA as a diagnostic tool The high-affinity binding of PNA oligomers has led to the development of applications as diagnostic probes for detecting genetic mutations9. Single base pair mutation analysis using PNA-directed PCR clamping: 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 technique54,55,56,57. This strategy 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 PCR54. 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 position54. PNAs as probe for nucleic acid biosensor: PNAs can be used as probe for sequence-specific biosensors2,9. This approach is based on immobilization of a PNA probe 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 transducer9. CONCLUSIONPeptide nucleic acids provide a novel class of compounds with wide biological potential. Their very specificinteractions with DNA and RNA and their chemical and biological stability make them promising both astherapeutic lead compounds and agents for diagnostic applications. The application of PNA as a genetictherapeutic 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 manyareas of chemistry, biology, and biotechnology. Recent efforts to provide further applications of this excitingnucleic acid analog include modifications of backbone and the development of novel baseanalogs36,49-58. REFERENCES* This article has been realized in the context of Piemonte region DIADI 2000 project1. 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(1993) Sequence specific double strand DNAcleavage by peptide nucleic acid (PNA) targeting using nuclease S1. Nucleic Acids Res. 21, 2103–2107 52. Lansdorp, P. M., Verwoerd, N. P., van de Rijke, F. M., Dragowska, V., Little, M. T., Dirks, R. W., Raap, A. K., and Tanke, H. J. (1996)Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 53. O rum, H., Nielsen, P. E., Jorgensen, M., Larsson, C., Stanley, C., and Koch, T. (1995) Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection. BioTechniques 19, 472–480 54. Orum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Research, Vol 21, Issue 23 5332-5336 55. Behn M, Schuermann M Simple and reliable factor V genotyping byPNA-mediated PCR clamping Thromb Haemost 1998, 79:773-77756. Murdock DG, Wallace DC PNA-mediated PCR clamping. Applications andmethods Methods Mol Biol. 2002;208:145-64. 57. Orum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C Single bese pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res1993, 21:5332-5336. Supplement to Chimica Oggi/CHEMISTRY TODAY Vol 23 nr 3 • Focus on Peptides & Amino Acids19SILVIA VELASCO,BARBARA CANEPA Dipartimento di genetica, biologia ebiochimica, Università di Torinoc/o Bioindustry Park del Canavese,10010 Colleretto Giacosa (TO)