Peptide nucleic acid (PNA) antisense effects in Escherichia coli

Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake, antisense PNAs may find applications as antimicrobial agents and as tools for microbial functional genomics.     

Antibiotic-free bacterial strain selection using antisense peptide nucleic acid

Antibiotics are widely useful in medicine, agriculture, and industrial fermentations. However, increasing problems with resistant strains call for restrained use and alternative strategies. Antisense peptide nucleic acids (PNAs) show potent bactericidal effects when targeted against the essential Escherichia coli acpP gene. Aside from attractive antimicrobial therapeutic possibilities for such antisense PNAs, we considered that they could be used as a substitute for antibiotics in bacterial strain selection. Here, treatment of a mixture of E. coli wild-type cells and cells carrying a binding-site altered copy of acpP (acpP-1) with anti-acpP PNA completely killed wild-type cells within 2 h, whereas cells carrying acpP-1 proliferated. Furthermore, electrotransformation of E. coli cells with the plasmid carrying acpP-1 followed by PNA selection gave rise to only true transformants. Unlike previous antibiotic-free selection strategies, this procedure does not require special growth environments or special host strains. Also, the PNA-selected cells grow at a near normal rate. The results open possibilities to use antisense PNAs for strain selection and construction in research and industrial application.     

Bactericidal antisense effects of peptide-PNA conjugates

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide-PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 microM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide-PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.     

Facile synthesis of peptide nucleic acids and peptide nucleic acid‐peptide conjugates on an automated peptide synthesizer

Peptide nucleic acids (PNAs) are DNA mimics with a neutral peptide backbone instead of the negatively charged sugar phosphates. PNAs exhibit several attractive features such as high chemical and thermal stability, resistance to enzymatic degradation, and stable binding to their RNA or DNA targets in a sequence‐specific manner. Therefore, they are widely used in molecular diagnosis of antisense‐targeted therapeutic drugs or probes and in pharmaceutical applications. However, the main hindrance to the effective use of PNAs is their poor uptake by cells as well as the difficult and laborious chemical synthesis. In order to achieve an efficient delivery of PNAs into cells, there are already many published reports of peptides being used for transport across the cell membrane. In this protocol, we describe the automated as well as cost‐effective semi‐automated synthesis of PNAs and PNA‐peptide constructs on an automated peptide synthesizer. The facile synthesis of PNAs will be helpful in generating PNA libraries usable, e.g. for high‐throughput screening in biomolecular studies. Efficient synthetic schemes, the automated procedure, the reduced consumption of costly reagents, and the high purity of the products are attractive features of the reported procedure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of Mycobacterium smegmatis gene expression and growth using antisense peptide nucleic acids

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.     

Peptide nucleic acids as therapeutic agents.

Peptide nucleic acids (PNAs) have been around for more than seven years and it was hoped, at their introduction, that they would quickly enter the fields of antisense and antigene technology and drug development. Despite their extremely favorable hybridization and stability properties, as well as the encouraging antisense and antigene activity of PNA in cell-free systems, progress has been slow and experiments on cells in culture and in animals have been lacking. Judging from the very promising results published within the past year, however, there is every reason to believe that both PNA antisense and, possibly, PNA antigene research will strongly pick up momentum again. Specifically, it has been demonstrated that certain peptide-PNA conjugates are taken up very efficiently by, at least some, eukaryotic cells and that antisense down regulation of target genes in nerve cells in culture is attainable using such PNA conjugates. Perhaps even more exciting is that antisense-compatible effects have been reported using PNAs injected into the brain of rats. Finally, it has been shown that the bacterium Escherichia coli is susceptible to antisense gene regulation using PNA.     

Efficient and isoform-selective inhibition of cellular gene expression by peptide nucleic acids

Peptide nucleic acids (PNAs) are a potentially powerful approach for the recognition of cellular mRNA and the inhibition of gene expression. Despite their promise, the rules for using antisense PNAs have remained obscure, and antisense PNAs have been used sparingly in research. Here we investigate the ability of PNAs to be effective antisense agents inside mammalian cells, to inhibit expression of human caveolin-1 (hCav-1), and to discriminate between its alpha and beta isoforms. Many human genes are expressed as isoforms. Isoforms may play different roles within a cell or within different tissues, and defining these roles is a challenge for functional genomics and drug discovery. PNAs targeted to the translation start codons for the alpha and beta isoforms inhibit expression of hCav-1. Inhibition is dependent on PNA length. The potency and duration of inhibition by PNAs are similar to inhibition of gene expression by short interferring RNA (siRNA). Expression of the alpha isoform can be blocked selectively by a PNA. Cell proliferation is halted by inhibition of expression of both hCav-1 isoforms, but not by inhibition of the alpha hCav-1 isoform alone. Efficient antisense inhibition and selective modulation of isoform expression suggest that PNAs are versatile tools for controlling gene expression and dissecting the roles of closely related protein variants. Potent inhibition by PNAs may supply a "knock down" technology that can complement and "cross-check" siRNA and other approaches to antisense gene inhibition that rely on oligomers with phosphate or phosphorothioate backbone linkages.     

The peptide nucleic acids (PNAs): "high-tech" probes for genetic and molecular cytogenetic investigations

The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acids analogs, in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by amine bonds and to which the nucleobases are fixed. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences, and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy and genome mapping. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic polymorphisms and mutations or capturing nucleic acids. Over the last few years, the use of PNAs has proven its powerful usefulness in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, spermatozoa as well as on isolated oocytes and blastomeres. Muticolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful tool for in situ chromosomal investigation.     

The use of peptide nucleic acids for in situ identification of human chromosomes.

The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acid analogues, based on their peptide-like backbone. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double-stranded DNA, the unique physicochemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy, genome mapping, and mutation detection. Over the past few years, PNAs have been shown to be powerful tools in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, and spermatozoa, as well as on isolated oocytes and blastomeres. Multicolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful complement to FISH for in situ chromosomal investigation.     

PNA/DNA interstrand cross-links from a modified PNA base upon photolysis or oxidative conditions

PNA/DNA interstrand cross-links (ICLs) were observed when peptide nucleic acids (PNAs) containing modified thymine derivatives were hybridized with the complementary or one-base mismatched DNA upon photolysis or treatments of oxidative agent. PNA/DNA ICL formation provides a useful method for biological applications such as antisense technologies or PNA chips.     

Delivery of antisense peptide nucleic acids (PNAs) to the cytosol by disulphide conjugation to a lipophilic cation

Peptide nucleic acids (PNAs) are effective antisense reagents that bind specific mRNAs preventing their translation. However, PNAs cannot cross cell membranes, hampering delivery to cells. To overcome this problem we made PNAs membrane-permeant by conjugation to the lipophilic triphenylphosphonium (TPP) cation through a disulphide bond. The TPP cation led to efficient PNA uptake into the cytoplasm where the disulphide bond was reduced, releasing the antisense PNA to block expression of its target gene. This method of directing PNAs into cells is a significant improvement on current procedures and will facilitate in vitro and pharmacological applications of PNAs.     

Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid ("umbrella") and cholesterol conjugates delivered by cationic lipids

Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid, exhibited low nanomolar activity (EC(50) ～ 25 nM). Excellent sequence specificity was retained, as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency, suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates, exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.     

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.     

Cell permeabilization and uptake of antisense peptide-peptide nucleic acid (PNA) into Escherichia coli.

Peptide nucleic acid (PNA) is a DNA mimic with promising properties for the development of antisense agents. Antisense PNAs targeted to Escherichia coli genes can specifically inhibit gene expression, and attachment of PNA to the cell-permeabilizing peptide KFFKFFKFFK dramatically improves antisense potency. The improved potency observed earlier was suggested to be due to better cell uptake; however, the uptake kinetics of standard or modified PNAs into bacteria had not been investigated. Here we monitored outer and inner membrane permeabilization by using chemical probes that normally are excluded from cells but can gain access at points where membrane integrity is disturbed. Membrane permeabilization was much more rapid in the presence of peptide-PNA conjugates relative to the free components used alone or in combination. Indeed, peptide-PNAs permeabilized E. coli nearly as quickly as antimicrobial peptides. Furthermore, as expected for outer membrane-active compounds, added MgCl(2) reduced cell-permeabilization. Concurrent monitoring of outer and inner membrane permeabilization indicated that passage across the outer membrane is rate-limiting for uptake. The enhanced cell-permeation properties of peptide-PNAs can explain their potent antisense activity, and the results indicate an unanticipated synergy between the peptide and PNA components.     

Cellular delivery of polyheteroaromate-peptide nucleic acid conjugates mediated by cationic lipids

In the search of facile and efficient methods for PNA cellular delivery, we have tested a series of PNA conjugates based on (hetero) aromatic, lipophilic compounds such as 9-aminoacridine, benzimidazoles, carbazole, anthraquinone, porphyrine, psoralen, pyrene, and phenyl-bis-benzimidazole ("Hoechst"). These chemically modified PNAs were delivered to cultured pLuc705HeLa cells mediated by cationic liposomes (LipofectAMINE or LiofectAMINE2000), and their nuclear delivery was inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. PNAs modified with 9-aminoacridine, "Hoechst", or acetyl-"Hoechst" showed highest antisense activities (while unmodified PNA failed to show any significant antisense activity). In particular, bis-acridine-conjugated PNA showed nearly 60% splicing correction at 250 nM concentration in combination with LipofectAMINE2000. Interestingly, relative differences between the derivatives were observed when LipofectAMINE was used as compared to LipofectAMINE2000, but in general the latter yielded the higher antisense activity. The most active modifications of these PNA constructs were further tested for antisense down-regulation of luciferase in p53R cells in order to evaluate the cytoplasmic activity (uptake) of the PNAs. A dose-dependent down regulation of luciferase was demonstrated also in this system. The PNA conjugated to acetyl-Hoechst caused a reduction of luciferase activity to less than 40% of the control at a concentration of 1 muM. These results indicate that conjugation of (hetero) polyaromatic compounds to PNA can dramatically improve liposome-mediated cellular delivery both to cytoplasm as well as to the nucleus. However, no clear structure/activity relations are apparent from the present results, except that both 9-aminoacridine and "Hoechst" are also nucleic acid binding ligands.     

Non-Watson-Crick interactions between PNA and DNA inhibit the ATPase activity of bacteriophage T4 Dda helicase

Peptide nucleic acid (PNA) is a DNA mimic in which the nucleobases are linked by an N-(2-aminoethyl) glycine backbone. Here we report that PNA can interact with single-stranded DNA (ssDNA) in a non-sequence-specific fashion. We observed that a 15mer PNA inhibited the ssDNA-stimulated ATPase activity of a bacteriophage T4 helicase, Dda. Surprisingly, when a fluorescein-labeled 15mer PNA was used in binding studies no interaction was observed between PNA and Dda. However, fluorescence polarization did reveal non-sequence-specific interactions between PNA and ssDNA. Thus, the inhibition of ATPase activity of Dda appears to result from depletion of the available ssDNA due to non-Watson–Crick binding of PNA to ssDNA. Inhibition of the ssDNA-stimulated ATPase activity was observed for several PNAs of varying length and sequence. To study the basis for this phenomenon, we examined self-aggregation by PNAs. The 15mer PNA readily self-aggregates to the point of precipitation. Since PNAs are hydrophobic, they aggregate more than DNA or RNA, making the study of this phenomenon essential for understanding the properties of PNA. Non-sequence-specific interactions between PNA and ssDNA were observed at moderate concentrations of PNA, suggesting that such interactions should be considered for antisense and antigene applications.     

Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.     

Antisense properties of duplex- and triplex-forming PNAs.

The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA.