Inhibition of gene expression inside cells by peptide nucleic acids: effect of mRNA target sequence, mismatched bases, and PNA length

Genome sequencing has revealed thousands of novel genes, placing renewed emphasis on chemical approaches for controlling gene expression. Antisense oligomers designed directly from the information generated by sequencing are one option for achieving this control. Here we explore the rules governing the inhibition of gene expression by peptide nucleic acids (PNAs) inside cells. PNAs are a DNA/RNA mimic in which the phosphate deoxyribose backbone has been replaced by uncharged linkages. Binding to complementary sequences is not hindered by electrostatic repulsion and is characterized by high rates of association and elevated affinities. Here we test the hypothesis that the favorable properties of PNAs offer advantages for recognition of mRNA and antisense inhibition of gene expression in vivo. We have targeted 27 PNAs to 18 different sites throughout the 5'-untranslated region (5'-UTR), start site, and coding regions of luciferase mRNA. PNAs were introduced into living cells in culture as PNA-DNA-lipid complexes, providing a convenient high throughput method for cellular delivery. We find that PNAs targeted to the terminus of the 5'-UTR are potent and sequence-specific antisense agents. PNAs fifteen to eighteen bases in length were optimal inhibitors. The introduction of one or two mismatches abolished inhibition, and complementary PNAs targeted to the sense strand were also inactive. In striking contrast to effective inhibition by PNAs directed to the terminal region, PNAs complementary to other sites within the 5'-UTR do not inhibit gene expression. We also observe no inhibition by PNAs complementary to the start site or rest of the coding region, nor do we detect inhibition by PNAs that are highly C/G rich and possess extremely high affinities for their target sequences. Our results suggest that PNAs can block binding of the translation machinery but are less able to block the progress of the ribosome along mRNA. The high specificity of antisense inhibition by PNAs emphasizes both the promise and the challenges for PNAs as antisense agents and provides general guidelines for using PNAs to probe the molecular recognition of biological targets inside cells.     

Strand invasion by mixed base PNAs and a PNA-peptide chimera

Peptide nucleic acid oligomers (PNAs) have a remarkable ability to invade duplex DNA at polypurine–polypyrimidine target sequences. Applications for PNAs in medicine and biotechnology would increase if the rules governing their hybridization to mixed base sequences were also clear. Here we describe hybridization of PNAs to mixed base sequences and demonstrate that simple chemical modifications can enhance recognition. Easily synthesized and readily soluble eight and 10 base PNAs bind to plasmid DNA at an inverted repeat that is likely to form a cruciform structure, providing convenient tags for creating PNA–plasmid complexes. PNAs also bind to mixed base sequences that cannot form cruciforms, suggesting that recognition is a general phenomenon. Rates of strand invasion are temperature dependent and can be enhanced by attaching PNAs to positively charged peptides. Our results support use of PNAs to access the information within duplex DNA and demonstrate that simple chemical modifications can make PNAs even more powerful agents for strand invasion. Simple strategies for enhancing strand invasion should facilitate the use of PNAs: (i) as biophysical probes of double-stranded DNA; (ii) to target promoters to control gene expression; and (iii) to direct sequence-specific mutagenesis.     

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.     

Intracellular uptake and inhibition of gene expression by PNAs and PNA-peptide conjugates

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.     

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.     

Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos

ABSTRACT: BACKGROUND: Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications. RESULTS: We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes. CONCLUSIONS: PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.     

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.     

Oligonucleotide-templated reactions based on Peptide Nucleic Acid (PNA) probes: Concept and biomedical applications

Sensing technologies based on Peptide Nucleic Acids (PNAs) and oligonucleotide-templated chemistry are perfectly suited for biomedical applications (e.g., diagnosis, prognosis and stratification of diseases) and could compete well with more traditional amplification technologies using expensive dual-labelled oligonucleotide probes. PNAs can be easily synthesised and functionalised, are more stable and are more responsive to point-mutations than their DNA counterpart. For these reasons, fluorogenic PNAs represent an interesting alternative to DNA-based molecular beacons for sensing applications in a cell-free environment, where cellular uptake is not required.     

Peptide nucleic acids specifically cause antigene effects in vivo by systemic injection

Peptide nucleic acids (PNAs) are uncharged DNA analogs that hybridize to complementary sequences with high affinity and stability. We previously showed that PNAs, after intraperitoneal injection into rats, are effective antisense compounds in vivo. The present study was designed to test whether PNAs also have antigene effects in vivo. The renin-angiotensin system is critical in the control of blood pressure. We designed and synthesized sense (antigene) PNAs to angiotensinogen, which is the precursor protein that leads to angiotensin I and II. Spontaneously hypertensive rats received intraperitoneal injections of either 20 mg/kg sense-angiotensinogen-PNA, mismatch-angiotensinogen PNA, or saline. Only the sense-angiotensinogen PNA treatment resulted in a significant decrease in plasma angiotensin I, systolic blood pressure, and liver and brain angiotensinogen mRNA levels. Thus, these results demonstrate on the molecular, protein, and physiological levels that antigene PNAs are effective in vivo upon systemic administration.     

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.     

Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.     

Liver cell specific targeting of peptide nucleic acid oligomers

Chimeric molecules consisting of peptide nucleic acid (PNA) and lactose have been synthesized to test the hypothesis that lactose moieties can promote cell-specific uptake of PNAs. We find that lactose modified PNAs rapidly enter liver-derived HepG2 cells while unmodified PNAs do not and that lactose modified PNAs can inhibit cellular telomerase.     

Russia's protected areas: a survey and identification of development problems

Natural habitat preservation, i.e. the creation and management of Protected Natural Areas (PNAs), is one of the most important forms of biodiversity conservation. The most widespread types of PNAs in Russia are Zakazniks (State Nature Refuges) and Natural Monuments, but unlike Zapovedniks (State Nature Reserves) these types of Russian PNAs are little-known to foreign ecologists. Thus the main attention of this article is given to the problems of Zakazniks and Natural Monuments while other types of Russian PNAs are mentioned briefly. In many regions of Russia, Zakazniks and Natural Monuments are considered to be the core components for the regional protection of biodiversity. Non-Governmental Organizations play an important role in the creation and management of PNAs. The recent sudden change of circumstances in Russia have given rise to many problems which threaten the existence of Zakazniks and Natural Monuments. Possible means of saving these PNAs include: (i) promoting the interest of local people in protecting biodiversity; and (ii) supporting local authorities, and public initiatives and regional programmes in the creation of local PNA networks.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.     

PNA-based antisense oligonucleotides for micrornas inhibition in the absence of a transfection reagent

MicroRNAs (miRNAs) are noncoding RNAs approximately 22 nucleotides in length that play a major role in the regulation of important biological processes, including cellular development, differentiation, and apoptosis. Antisense oligonucleotides against miRNAs are useful tools for studying the biological mechanisms and therapeutic targets of miRNAs. Various antisense oligonucleotides chemistries, including peptide nucleic acids (PNAs), have been developed to enhance nuclease-resistance and affinity and specificity for miRNA targets. PNAs have a greater specificity and affinity for DNA and RNA than do natural nucleic acids, and they are resistant to nucleases-an essential property of an miRNA inhibitor that will be exposed to cellular nucleases. However, the main limiting factor in the use of PNAs is their reduced penetration into cells. Recently, several cell-penetrating peptides (CPPs) have been investigated as a means to overcome the limited penetration of PNAs. Here, we evaluated the ability of 11 CPPs to transport PNAs inside cells in the absence of transfection reagents and then investigated the ability of these CPPs to inhibit miRNAs. Of the 11 CPPs tested, Tat-modified-conjugated PNA showed the most effective penetration into cells in the absence of transfection reagents and most effectively inhibited miRNAs. Our data demonstrate that Tat-modified-conjugated CPP is the most suitable for supporting PNA-mediated miRNA inhibition.     

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.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Summary Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.