Unmodified Peptide Nucleic Acids Research Articles

Inhibition of Neomycin Phosphorotransferase Expression in Entamoeba histolytica with Antisense Peptide Nucleic Acid (PNA) Oligomers

PNA are DNA analogs in which the entire phosphate-sugar backbone has been replaced by an isomorphic pseudopeptidic chain to which the four bases are linked. They have been shown to bind complementary natural polynucleotide sequences with higher affinity than their natural counterparts, partially due to the high flexibility and absence of charge of the artificial backbone. Furthermore, their artificial structure is not recognized by proteases and nucleases, making PNA exceptionally stable in biological environments (1). This consideration is of particular relevance to Entamoeba histolytica , as it possesses numerous hydrolases, some membrane bound. PNA oligomers have been used in the last few years as antisense and antigene agents in a variety of cell-free and cellular systems, showing that they can effectively and selectively downregulate gene expression at the translational or transcriptional levels when used at micromolar concentrations (2). The difficulties inherent in the cultivation of E. histolytica trophozoites and the lack of procedures for deletion of genes have made it very difficult to perform classical genetic studies aimed at the characterization of phenotypes resulting from gene inactivation. Only recently, antisense strategies using plasmids transcribing antisense mRNAs have been successfully used, allowing the downregulation of specific genes and the analysis of resulting phenotypes (3). In this study, we report the use of PNA oligomers as antisense agents for effective and specific downregulation of expression of the bacterial gene neomycin phosphorotransferase (NPT) in E. histolytica in culture, with results comparable to antisense strategies recently reported. In contrast to other cellular types tested, notably mammalian cells, E. histolytica is permeable to unmodified PNA, and inhibition of gene expression is significant and easily achieved in a dose-dependent manner, saving the work of cloning, transfection, and selection procedures needed for plasmid-mediated antisense inhibition.

In Vitro Membrane Penetration of Modified Peptide Nucleic Acid (PNA)

Efficient cellular uptake is crucial for the success of any drug directed towards targets inside cells. Peptide nucleic acid (PNA), a DNA analog with a promising potential as a gene-directed drug, has been shown to display slow membrane penetration in cell cultures. We here used liposomes as an in vitro model of cell membranes to investigate the effect on penetration of a PNA molecule colvalently modified with a lipophilic group, an adamantyl moiety. The adamantyl attachment was found to increase the membrane-penetration rate of PNA three-fold, as compared to corresponding unmodified PNA. From the penetration behaviour of a number of small and large molecules we could conclude that passive diffusion is the mechanism for liposome-membrane passage. Flow linear dichroism (LD) of the modified PNA in presence of rod-shaped micelles, together with octanol-water distribution experiments, showed that the adamantyl-modified PNA is amphiphilic; the driving force behind the observed increased membrane-penetration rate appears to be an accumulation of the PNA in the lipid double layer.

Peptide nucleic acids (PNA) derived from N-(N-methylaminoethyl)glycine. Synthesis, hybridization and structural properties

Backbone N-methylated peptide nucleic acids (PNAs) containing the four nucleobases adenine, cytosine, guanine and thymine were synthesized via solid phase peptide oligomerization. The oligomers bind to their complementary target with a thermal stability that is 1.5–4.5°C lower per '‘N-methyl nucleobase unit’' [dependent on the number and position(s) of the N-methyl] than that of unmodified PNA. However, even fully N-methyl modified PNAs bind as efficiently to DNA or RNA targets as DNA itself. Furthermore, the hybridization efficiency per N-methyl unit in a PNA decreased with increasing N-methyl content, and the effect was more pronounced when the N-methyl backbone units are present in the Hoogsteen versus the Watson–Crick strand in (PNA)2-DNA triplexes. Interestingly, CD spectral analyses indicate that 30% (3 out of ten) substitution with N-methyl nucleobases did not alter the structure of PNA-DNA (or RNA) duplexes or (PNA)2-DNA triplexes, and likewise CD spectroscopy and X-ray crystallography showed no major structural differences between N-methylated (30%) and unmodified PNA-PNA duplexes. However, PNA-DNA duplexes as well as triplexes adopted a different conformation when formed with all-N-methyl PNAs.

Phospholipid membrane permeability of peptide nucleic acid

Phospholipid vesicles (liposomes) as membrane models have been used to study the penetration properties of peptide nucleic acid (PNA), a new DNA analog in which the nucleobases are attached to a pseudo-peptide backbone. The liposomes were characterised by carboxyfluorescein efflux, light-scattering and cryogenic transmission electron microscopy. The liposome structure was found not to be affected by the incorporation of PNA or an oligonucleotide. Two 10-mer fluorescein-labelled PNAs were found to have low efflux rates (half-times of 5.5 and 11 days), comparable to a 10-mer oligonucleotide (half-time of 7 days). We conclude that passive diffusion of unmodified PNA is not an effective way of transport into biological cells.

Structure-Activity Studies of the Binding of Modified Peptide Nucleic Acids (PNAs) to DNA

Peptide nucleic acid (PNA) oligomers where one of the repeating backbone units is extended with a methylene group to either N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine were prepared. Alternatively, the linker to the nucleobase was extended from methylenecarbonyl to ethylenecarbonyl. The thermal stability of the hybrids between these PNA oligomers and complementary DNA oligonucleotides was significantly lower than that of the corresponding complexes involving unmodified PNA. However, the sequence selectivity was retained. Thymidyl decamers with all N-(2-aminoethyl)-beta-alanine or N-(3-aminopropyl)glycine backbones were prepared and shown to be unable to hybridize to the complementary (dA)(10) oligonucleotides, whereas a PNA decamer containing only ethylenecarbonyl linkers between the nucleobases and the N-(2-aminoethyl)glycine backbone showed weak but sequence-specific affinity for complementary DNA. All hybrids involving homopyrimidine PNA oligomers exhibited (PNA)(2)/DNA stoichiometry, whereas mixed-sequence PNA oligomers formed PNA/DNA duplexes. The preferred binding direction between the modified PNA and DNA in the duplex motifs was antiparallel, as previously reported for complexes involving unmodified PNA.