Information transfer from peptide nucleic acids to RNA by template-directed syntheses.

Peptide nucleic acids (PNAs) are uncharged analogs of DNA and RNA in which the ribose-phosphate backbone is substituted by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. A PNA C10 oligomer has been shown to act as template for efficient formation of oligoguanylates from activated guanosine ribonucleotides. In a previous paper we used heterosequences of DNA as templates in sequence-dependent polymerization of PNA dimers. In this paper we show that information can be transferred from PNA to RNA. We describe the reactions of activated mononucleotides on heterosequences of PNA. Adenylic, cytidylic and guanylic acids were incorporated into the products opposite their complement on PNA, although less efficiently than on DNA templates.     

Control of electron transfer in DNA by peptide nucleic acids (PNA).

The one-electron oxidation of PNA-DNA hybrid containing G-triplet sequence was examined. In DNA duplex G-triplet was selectively cleaved by oxidation, whereas in PNA-DNA hybrid cleavage efficiency was extremely lowered. These result suggested that cleavage efficiency of PNA-DNA hybrid was different from that of B-form DNA duplex.     

Transcription and reverse transcription of artificial nucleic acids involving backbone modification by template-directed DNA polymerase reactions

Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been replaced with an amide-type linker [–CH₂CONH–] or an amine-type linker [–CH₂CH₂NH–] were synthesized to investigate the effect of these backbone modifications on polymerase reactions. In addition, a triphosphate analogue of thymidine dinucleotide with the amide-type linker was synthesized and enzymatic insertion of the amide linkage into ODN was attempted using this analogue for the polymerase reaction. Primer extension reactions using three types of thermostable DNA polymerases, KOD(exo-), Vent(exo-) and Taq were performed for the assays. Analysis of these data indicate that (i) the polymerase reaction tends to be affected much more by insertion of the cationic flexible amine-type linker than by insertion of the neutral rigid amide-type linker; (ii) the backbone modification has a greater effect on the polymerase reaction when it is adjacent to the 3′-end of a primer as the elongation terminus than when it is on the template, as well as in base or sugar modification; (iii) although the modified linker in the modified DNA template is passed beyond by the polymerase, it still affects the extension reaction several bases downstream from its location; (iv) the modified linker in the template, in some cases, also affects the extension reaction upstream from its location; (v) further improvement of the chemical structure is required for dinucleotide-mimic incorporation.     

Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (T_m) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the T_m of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a T_m around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9–DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.     

Strand invasion by DNA-peptide conjugates and peptide nucleic acids.

Peptide nucleic acids (PNAs) and conjugates between oligonucleotides and cationic peptides possess superior potential for strand invasion at complementary sequences. We discovered that oligonucleotide-peptide conjugates and PNAs fall into three classes based on their hybridization efficiency; i) those complementary to inverted repeats within AT-rich region hybridize with highest efficiency; ii) those complementary to areas adjacent to inverted repeats or near AT-rich regions hybridize with moderate efficiency; and iii) those complementary to other regions do not detectably hybridize. The correlations between oligomer chemistry, DNA target sequence, and hybridization efficiency that we report here have important implications for the recognition of duplex DNA.     

Sequence-specific targeting of duplex DNA by peptide nucleic acids via triplex strand invasion

Because of a set of exceptional chemical, physical, and biological properties, polyamide or peptide nucleic acids (PNAs) hold a distinctive position among various synthetic ligands designed for DNA-targeting purposes. Cationic pyrimidine PNAs (cpyPNAs) represent a special group of PNAs, which effectively form strand invasion triplexes with double-stranded DNA (dsDNA) also known as P-loops. Extraordinary stability of the invasion triplexes and high sequence specificity of their formation combined with local opening of the DNA double helix within the P-loops make these complexes very attractive for sequence-specific manipulation with dsDNA. Important for applications is the fact that the discrimination between correct and mismatched binding sites in dsDNA by cpyPNAs is a nonequilibrium, kinetically controlled process. Therefore, a careful choice of experimental conditions that are optimal for the kinetic discrimination of correct versus mismatched cpyPNA binding is crucial for sequence-specific recognition of dsDNA by cpyPNAs. The experimental and theoretical data presented make it possible to select those solution parameters and cpyPNA constructions that are most favorable for sequence specificity without compromising the affinity of dsDNA targeting.     

Kinetic analysis of specificity of duplex DNA targeting by homopyrimidine peptide nucleic acids

A simple theoretical analysis shows that specificity of double-stranded DNA (dsDNA) targeting by homopyrimidine peptide nucleic acids (hpyPNAs) is a kinetically controlled phenomenon. Our computations give the optimum conditions for sequence-specific targeting of dsDNA by hpyPNAs. The analysis shows that, in agreement with the available experimental data, kinetic factors play a crucial role in the selective targeting of dsDNA by hpyPNAs. The selectivity may be completely lost if PNA concentration is too high and/or during prolonged incubation of dsDNA with PNA. However, quantitative estimations show that the experimentally observed differences in the kinetic constants for hpyPNA binding with the correct and mismatched DNA sites are sufficient for sequence-specific targeting of long genomic DNA by hpyPNAs with a high yield under appropriate experimental conditions. Differential dissociation of hpyPNA/dsDNA complexes is shown to enhance the selectivity of DNA targeting by PNA.     

Applications of peptide nucleic acids

Several exciting new developments in the applications of the DNA mimic peptide nucleic acid (PNA) have been published recently. A possible breakthrough may have come in efforts to develop PNA into gene therapeutic drugs. In eukaryotic systems, antisense activity of PNAs (as peptide conjugates) has been reported in nerve cells and even in rats upon injection into the brain, and antisense activity has also been demonstrated in Escherichia coli. PNA hybridization technology has developed rapidly within in situ hybridization, and exciting new methods based on MALDI-TOF detection have also been presented.     

Synthesis and DNA binding properties of terminally modified peptide nucleic acids

PNAs with terminal modifications of varying structure and charge were synthesized and their binding to DNA was studied. A variation in thermal stability of 19. 8 degrees C has been observed between the least and the most stable PNA-DNA duplexes. The most stable duplex melts 7.7 degrees C higher than the duplex of the corresponding non-modified PNA and complementary DNA. It has been shown that sequence fidelity of the PNA conjugate having the highest DNA affinity is significantly better than that of non-modified PNA. The results obtained can be used for the design of PNA probes, whose binding to DNA is sequence independent.     

A DNA biosensor based on peptide nucleic acids on gold surfaces

We present a DNA biosensor based on self-assembled monolayers (SAMs) of thiol-derivatized peptide nucleic acid (PNA) molecules adsorbed on gold surfaces. Previous works have shown that PNA molecules at an optimal concentration can be self-assembled with their molecular axes normal to the surface. In such structural configuration BioSAMs of PNAs maintain their capability for recognizing complementary DNA. We describe the combined use of PM-RAIRS and synchrotron radiation XPS for the detection and spectroscopic characterization of PNA-DNA hybridization process on gold surfaces. RAIRS and XPS are powerful techniques for surface characterization and molecular detection, which do not require a fluorescence labeling of the target. We present a characterization of the spectroscopic IR and XPS features, some of them associated to the phosphate groups of the DNA backbone, as an unambiguous signature of the PNA-DNA heteroduplex formation. The N(1s) XPS core level peak after DNA hybridization is decomposed in curves components, and every component assigned to different chemical species. Therefore, the results obtained by means of two complementary structural characterization techniques encourage the use of PNA-based biosensors for the detection of DNA molecules on natural samples.     

Modulation of remote DNA oxidation by hybridization with peptide nucleic acids (PNA)

We have examined the efficiency of DNA photooxidation in DNA/PNA duplex and DNA/(PNA)(2) triplex for the first time. DNA/PNA duplex was cleaved at GG steps by external riboflavin with high efficiency like specific GG cleavage in DNA/DNA duplex. However, the 5'G selectivity of the GG oxidation in DNA/PNA duplex was much lower than that observed in DNA/DNA duplex. Remote DNA oxidation of oxidant-tethered DNA/PNA duplex was considerably suppressed. In contrast, the formation of DNA/(PNA)(2) triplex by hybridization with two PNA strands completely inhibited the remote GG oxidation, indicating that PNA acts as an inhibition for remote oxidative DNA damage.     

Conformationally restricted chiral peptide nucleic acids derived from azetidines

The initial experiments towards the chemical synthesis of conformationally rigid peptide nucleic acid analogues with azetidine moieties have been described.     

Recognition of double-stranded RNA by guanidine-modified peptide nucleic acids

Double-helical RNA has become an attractive target for molecular recognition because many noncoding RNAs play important roles in the control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double-helical RNA via formation of a triple helix. Herein, we tested if the molecular recognition of RNA could be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-workers that the guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple-helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double-helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from d-arginine recognized the transactivation response element of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNA and the purine-rich strand of the bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex noncoding RNAs.     

Induction of splice correction by cell-penetrating peptide nucleic acids

BACKGROUND: Directing splicing using oligonucleotides constitutes a promising therapeutic tool for a variety of diseases such as beta-thalassemia, cystic fibrosis, and certain cancers. The rationale is to block aberrant splice sites, thus directing the splicing of the pre-mRNA towards the desired protein product. One of the difficulties in this setup is the poor bioavailability of oligonucleotides, as the most frequently used transfection agents are unsuitable for in vivo use. Here we present splice-correcting peptide nucleic acids (PNAs), tethered to a variety of cell-penetrating peptides (CPPs), evaluating their mechanism of uptake and ability to correct aberrant splicing. METHODS: HeLa cells stably expressing luciferase containing an aberrant splice site were used. A previously described PNA sequence, capable of correcting the aberrant splicing, was conjugated to the CPPs, Tat, penetratin and transportan, via a disulfide bridge. The ability of the CPP-PNA conjugates to correct splicing was measured, and membrane disturbance and cell viability were evaluated using LDH leakage and WST-1 assays. Lysosomotropic agents, inhibition of endocytosis at 4 degrees C and confocal microscopy were used to investigate the importance of endocytosis in the uptake of the cell-penetrating PNAs. RESULTS: All the three CPPs were able to promote PNA translocation across the plasma membrane and induce splice correction. Transportan (TP) was the most potent vector and significantly restored splicing in a concentration-dependent manner. Interestingly, TP also rendered a concentration-dependent splice correction in serum, in contrast to Tat and penetratin. Addition of the lysosomotrophic agent chloroquine increases the splice correction efficacy of the CPP-PNA conjugates up to 4-fold, which together with experiments at 4 degrees C and the visual information from confocal microscopy, indicate that the mechanism of uptake responsible for internalization of CPP-PNA conjugates is mainly endocytic. Finally, co-localization studies with dextran further indicate that conjugates, at least in the case of TP, internalize via endocytosis and in particular macropinocytosis. CONCLUSIONS: These data demonstrate that CPPs can be used for the delivery of splice-correcting PNAs, with potential to be used as a therapeutic approach for regulating splicing in a variety of diseases. Transportan presents itself as the overall most suitable vector in this study, generating the most efficient conjugates for splice correction.     

Blockade of plasmid replication mediated by peptide nucleic acids

Because peptide nucleic acids (PNAs) are capable of blocking amplification of deoxyribonucleic acid (DNA) by Taq DNA polymerase in vitro, we postulated that PNAs might be able to block replication in vivo. To explore this possibility, we assessed the ability of PNA to specifically block the replication of pUC19 plasmids by allowing a PNA, directed against segments of the Amp ^r sequence to bind to pUC19 prior to electroporation into Escherichia coli, strain DH10B. Colonies produced by this maneuver not only remained sensitive to ampicillin but were also incapable of blue color production on X-gal-containing media, thus demonstrating true blockade of pUC19 replication, rather than antisense activity. The ability of the PNA to prevent pUC19 replication in these experiments was shown to be dose related. Attempts to prevent the replication of E. coli using a PNA directed against a portion of the lac Z sequence found within the bacterial genome were not uniformly successful. Subsequent experiments showed that the electroporated PNA did not consistently enter a sufficient number of cells for an effect to be demonstrated in the assays used. Nonetheless, this is the first demonstration of in vivo complete replication blockade by a PNA and opens up the potential for new forms of specific antibiosis in both prokaryotic and eukaryotic cells.     

Solid-Phase synthesis of peptide nucleic acids

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH₂, indicated an average yield per synthetic cycle of 97.1%. N¹-benzyloxycarbonyl-N6³-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the ‘low–high’ trifluoromethanesulphonic acid procedure.