Applications Of Peptide Nucleic Acids Research Articles

Efficient cell penetration and delivery of peptide nucleic acids by an argininocalix[4]arene

The application of Peptide Nucleic Acids (PNAs), mimics of DNA lacking the sugar-phosphate backbone, for antisense/anti-gene therapy and gene editing is limited by their low uptake by cells. Currently, no simple and efficient delivery systems and methods are available to solve this open issue. One of the most promising approach is the modification of the PNA structure through the covalent linkage of poliarginine tails, but this means that every PNA intended to be internalized must be modified. Herein we report the results relative to the delivery ability of a macrocyclic multivalent tetraargininocalix[4]arene (1) used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity and ease preparation of the transfection formulation, simply attained by mixing PNA and calixarene, candidate this vector as universal delivery system for this class of nucleic acid analogues.

Potential of Peptide Nucleic Acids in Future Therapeutic Applications.

Peptide nucleic acids (PNA) are synthetic analog of DNA with a repeating N-(2-aminoethyl)-glycine peptide backbone connected to purine and pyrimidine nucleobases via a linker. Considering the unique properties of PNA, including resistance to enzymatic digestion, higher biostability combined with great hybridization affinity toward DNA and RNA, it has attracted great attention toward PNA- based technology as a promising approach for gene alteration. However, an important challenge in utilizing PNA is poor intracellular uptake. Therefore, some strategies have been developed to enhance the delivery of PNA in order to reach cognate site. Although PNAs primarily demonstrated to act as an antisense and antigene agents for inhibition of transcription and translation of target genes, more therapeutic applications such as splicing modulation and gene editing are also used to produce specific genome modifications. Hence, several approaches based on PNAs technology have been designed for these purposes. This review briefly presents the properties and characteristics of PNA as well as different gene modulation mechanisms. Thereafter, current status of successful therapeutic applications of PNA as gene therapeutic intervention in different research areas with special interest in medical application in particular, anti-cancer therapy are discussed. Then it focuses on possible use of PNA as anti-mir agent and PNA-based strategies against clinically important bacteria.

Hydrophilic and Cell-Penetrable Pyrrolidinyl Peptide Nucleic Acid via Post-synthetic Modification with Hydrophilic Side Chains

Peptide nucleic acid (PNA) is a nucleic acid mimic in which the deoxyribose-phosphate was replaced by a peptide-like backbone. The absence of negative charge in the PNA backbone leads to several unique behaviors including a stronger binding and salt independency of the PNA-DNA duplex stability. However, PNA possesses poor aqueous solubility and cannot directly penetrate cell membranes. These are major obstacles that limit in vivo applications of PNA. In previous strategies, the PNA can be conjugated to macromolecular carriers or modified with positively charged side chains such as guanidinium groups to improve the aqueous solubility and cell permeability. In general, a preformed modified PNA monomer was required. In this study, a new approach for post-synthetic modification of PNA backbone with one or more hydrophilic groups was proposed. The PNA used in this study was the conformationally constrained pyrrolidinyl PNA with prolyl-2-aminocyclopentanecarboxylic acid dipeptide backbone (acpcPNA) that shows several advantages over the conventional PNA. The aldehyde modifiers carrying different linkers (alkylene and oligo(ethylene glycol)) and end groups (-OH, -NH2, and guanidinium) were synthesized and attached to the backbone of modified acpcPNA by reductive alkylation. The hybrids between the modified acpcPNAs and DNA exhibited comparable or superior thermal stability with base-pairing specificity similar to those of unmodified acpcPNA. Moreover, the modified apcPNAs also showed the improvement of aqueous solubility (10-20 folds compared to unmodified PNA) and readily penetrate cell membranes without requiring any special delivery agents. This study not only demonstrates the practicality of the proposed post-synthetic modification approach for PNA modification, which could be readily applied to other systems, but also opens up opportunities for using pyrrolidinyl PNA in various applications such as intracellular RNA sensing, specific gene detection, and antisense and antigene therapy.

Molecular self-assembly using peptide nucleic acids.

Peptide nucleic acids (PNAs) are extensively studied for the control of genetic expression since their design in the 1990s. However, the application of PNAs in nanotechnology is much more recent. PNAs share the specific base-pair recognition characteristic of DNA together with material-like properties of polyamides, both proteins and synthetic polymers, such as Kevlar and Nylon. The first application of PNA was in the form of PNA-amphiphiles, resulting in the formation of either lipid integrated structures, hydrogels or fibrillary assemblies. Heteroduplex DNA-PNA assemblies allow the formation of hybrid structures with higher stability as compared with pure DNA. A systematic screen for minimal PNA building blocks resulted in the identification of guanine-containing di-PNA assemblies and protected guanine-PNA monomer spheres showing unique optical properties. Finally, the co-assembly of PNA with thymine-like three-faced cyanuric acid allowed the assembly of poly-adenine PNA into fibers. In summary, we believe that PNAs represent a new and important family of building blocks which converges the advantages of both DNA- and peptide-nanotechnologies.

Diagnostic and therapeutic applications of peptide nucleic acid in medical microbiology

Diagnostic and therapeutic applications of peptide nucleic acid in medical microbiology

Recognition Mechanisms and Applications of Peptide Nucleic Acids Targeting Double-stranded DNA.

Targeting double-stranded DNA (dsDNA) with high affinity and specificity has become a hot topic in biochemistry and molecular biology research. Gene diagnosis and therapy, DNA manipulation, and gene expression regulation could be achieved based on certain recognition principles through specific interactions between dsDNA and natural nucleotides or synthesized ligands. Some ligands (e.g., peptide nucleic acids (PNAs), triple helix-forming oligonucleotides (TFOs), oligopolyamides, and zinc-finger peptides) show sufficient affinity and specificity for targeting dsDNA sequences. PNA can simply synthesize and recognize dsDNA without sequence limitation under physiological conditions. This paper provides a review on the recognition mechanisms, influencing factors, and applications of the four recognition modes of PNA targeting dsDNA. These modes include triplex invasion, triplex binding, duplex invasion, and double duplex invasion. This paper also discusses the challenges to be addressed by future research to fully explore the potential of PNA probe design for specific dsDNA recognition.

Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies.

Self-assembled polymer/porous silicon nanocomposites overcome intracellular and systemic barriers for in vivo application of peptide nucleic acid (PNA) anti-microRNA therapeutics. Porous silicon (PSi) is leveraged as a biodegradable scaffold with high drug-cargo-loading capacity. Functionalization with a diblock polymer improves PSi nanoparticle colloidal stability, in vivo pharmacokinetics, and intracellular bioavailability through endosomal escape, enabling PNA to inhibit miR-122 in vivo.

Delivery of cell-penetrating peptide-peptide nucleic acid conjugates by assembly on an oligonucleotide scaffold.

Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.

BSA as additive: A simple strategy for practical applications of PNA in bioanalysis.

Application of peptide nucleic acid (PNA) in bioanalysis has been limited due to its nonspecific adsorption onto hydrophobic surface in spite of favorable properties such as higher chemical/biological stability, specificity and binding affinity towards target nucleic acids compared to natural nucleic acid probes. Herein, we employed BSA in PNA application to enhance the stability of PNA in hydrophobic containers and improve the sensing performance of the DNA sensor based on graphene oxide (GO) and PNA. Addition of 0.01% BSA in a PNA solution effectively prevented the adsorption of PNA on hydrophobic surface and increased the portion of the effective PNA strands for target binding without interfering duplex formation with a complementary target sequence. In the GO based biosensor using PNA, BSA interrupted the unfavorable adsorption of PNA/DNA duplex on GO surface, while allowing the adsorption of ssPNA, resulting in improvement of the performance of the DNA sensor system by reducing the detection limit by 90-folds.

A Review of Peptide Nucleic Acid

Peptide Nucleic Acid (PNA) is a nucleobase oligomer in which the whole backbone is mainly replaced by N-(2- aminoethyl) glycine units. PNA is considered as DNA with a neutral peptide backbone due to negative charged sugar–phosphate backbone. It is chemically stable and resistant to hydrolytic cleavage. PNA can be categorized specific sequences of DNA and RNA according to Watson–Crick hydrogen bonding structure. Hybridization process showed high thermal stability and unique ionic strength effects. It is formed a stable PNA/DNA/PNA triplex with a looped-out DNA strand. PNA hybridization technology is promptly developed within in situ hybridization. In our review paper was elaborated the PNA superior hybridization characteristics, importance’s of PNA and major applications of PNA in the diagnostic and pharmaceutical fields. And also PNA could be replaced DNA in uses as a probe for many investigation purposes. PNAs antisense activities have found in nerve cells and even in rats upon injection into the brain, and in Escherichia coli.

Molecular properties and medical applications of peptide nucleic acids.

Peptide Nucleic Acids (PNAs) are molecules combining structural features of proteins and nucleic acid. They resemble DNA or RNA by forming helical polyamides containing nitrogen bases attached to the backbone consisting of N-(2-aminoethyl)-glycine monomers, which mimics the alternating ribose-phosphodiester-backbone of a nucleic acid. Because PNAs bind exceptionally strong to complementary DNA or RNA sequences obeying Watson-Crick base paring, they became attractive candidates for antisense and antigen therapies. PNAs are also being tested as novel antibiotics, gene-activating agents, and as molecular probes for FISH and imaging or biosensors used in diagnostics. Although PNAs offer many exiting medical applications, improving their cellular uptake and developing specific delivery strategies is crucial for a successful entry in the clinic in the near future.

Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene

Peptide nucleic acids (PNAs) have gained much interest as molecular recognition tools in biology, medicine and chemistry. This is due to high hybridization efficiency to complimentary oligonucleotides and stability of the duplexes with RNA or DNA. We have synthesized 15/16-mer PNA probes to detect the HER2 mRNA. The performance of these probes to detect the HER2 target was evaluated by fluorescence imaging and fluorescence bead assays. The PNA probes have sufficiently discriminated between the wild type HER2 target and the mutant target with single base mismatches. Furthermore, the probes exhibited excellent linear concentration dependence between 0.4 to 400 fmol for the target gene. The results demonstrate potential application of PNAs as diagnostic probes with high specificity for quantitative measurements of amplifications or over-expressions of oncogenes.

Facilitating Mismatch Discrimination by Surface-Affixed PNA Probes via Ionic Regulation

There has been a search for alternative nucleic acids that can be more effectively used in nucleic acid detection technologies compared to the DNA probes. Peptide nucleic acid (PNA), which contains a non-ionic peptidic backbone, offers such possibilities since it is nuclease-resistant, it binds to DNA with high affinity, and it can be readily self-assembled onto solid substrates, e.g., gold(111), with a molecular backbone orientation away from the substrate. Although application of PNA as a sensor probe has been exemplified, so far there is little or no account of the ionic modulation of single base mismatch discrimination capacity of surface-tethered PNA probes. Herein, we report "on-surface" melting temperatures of PNA-DNA duplexes formed on gold(111) surface, as obtained from fluorescence measurements. We show that surface-tethered PNA forms a stabler duplex than DNA, and is more effective in single base mismatch discrimination than DNA. Importantly, although PNA backbone is non-ionic, variation in the ionic components in hybridization buffer, i.e., varying concentration of monovalent sodium ion, and the nature of anion and the cation, exhibits clear effects on the mismatch discrimination capacity of PNA probes. In general, with decreasing cation concentration, PNA-DNA duplexes are stabilized and mismatch discrimination capacity of the PNA probes is enhanced. The stabilizing/destabilizing effects of anions are found to follow the Hofmeister series, emphasizing the importance of hydrophobic interaction between nucleobases for stability of the PNA-DNA duplexes. Interestingly, the nature of ionic dependence of "on-surface" mismatch detection ability of PNA probes differs significantly from the "solution" behavior of these probes.

Application of peptide nucleic acid (PNA)-PCR clamping technique to investigate the community structures of rhizobacteria associated with plant roots

The contamination of plant organelle (mitochondria and plastid) genes in the DNA extraction step becomes a major drawback in investigating the community structures of bacteria associated with plant samples. This is because organelle small subunit ribosomal RNA (SSU rRNA) genes are easily amplified by polymerase chain reaction (PCR) with a set of universal primers for bacteria. To suppress the PCR amplification of the organelle SSU rRNA genes, a peptide nucleic acid (PNA)-PCR clamping technique was applied for selective amplification of bacterial SSU rRNA genes. The PNA oligomers, which had sequences that were complementary to mitochondria and plastid SSU rRNA genes, were designed to overlap the region in the 1492r primer-binding site. PCR with the PNA oligomers significantly suppressed the amplification of the organelle SSU rRNA genes from spinach and cucumber roots. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that the conventional amplification without PNA oligomers generated the predominant T-RFLP fragments derived from mitochondria and plastids, whereas there was little detection of the rhizobacterial fragments. In contrast, several other T-RFLP fragments derived from rhizobacteria were detected in the products amplified with PNA oligomers, thereby enabling us to differentiate the community structures in spinach and cucumber roots. Thus, application of PNA-PCR clamping was considered to be effective and is a useful technique to amplify the rhizobacterial SSU rRNA genes from selectively extracted DNA containing plant mitochondria and plastid genes.

Applications of peptide nucleic acids (PNAs) and locked nucleic acids (LNAs) in biosensor development

Nucleic acid biosensors have a growing number of applications in genetics and biomedicine. This contribution is a critical review of the current state of the art concerning the use of nucleic acid analogues, in particular peptide nucleic acids (PNA) and locked nucleic acids (LNA), for the development of high-performance affinity biosensors. Both PNA and LNA have outstanding affinity for natural nucleic acids, and the destabilizing effect of base mismatches in PNA- or LNA-containing heterodimers is much higher than in double-stranded DNA or RNA. Therefore, PNA- and LNA-based biosensors have unprecedented sensitivity and specificity, with special applicability in DNA genotyping. Herein, the most relevant PNA- and LNA-based biosensors are presented, and their advantages and their current limitations are discussed. Some of the reviewed technology, while promising, still needs to bridge the gap between experimental status and the harder reality of biotechnological or biomedical applications.

Synthesis and characterization of conformationally preorganized, (R)-diethylene glycol-containing γ-peptide nucleic acids with superior hybridization properties and water solubility.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol ("miniPEG", R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure (R-MP)γPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine. Once synthesized, (R-MP)γPNA oligomers are preorganized into a right-handed helix, hybridize to DNA and RNA with greater affinity and sequence selectivity, and are more water soluble and less aggregating than the parental PNA oligomers. The results presented herein have important implications for the future design and application of PNA in biology, biotechnology, and medicine, as well as in other disciplines, including drug discovery and molecular engineering.

Application of peptide nucleic acids containing azobenzene self-assembled electrochemical biosensors in detecting DNA sequences

Hybridization of peptide nucleic acids probe containing azobenzene (NH2-TNT4, N-PNAs) with DNA was performed by covalently immobilizing of NH2-TNT4 in sequence on the 3-mercaptopropionic acid self-assembled monolayer modified gold electrode with the helps of N-(3-dimethylaminopropy1)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the hybrid was coded as N-PNAs/DNA. Using [Fe(CN)6]4−/3− (1:1) as the electrochemical indicator, the electrochemical properties of the N-PNAs self-assembled monolayer (N-PNAs-SAMs) and N-PNAs/DNA hybridization system under the conditions of before and after UV light irradiation were characterized with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectra (EIS). Results showed that the redox currents decreased with the increase of irradiation time, suggesting that the ability of the charge transfer on the electrode surface was weakened and the conformation of hybrid system had been changed, and the control of PNAs/DNA hybridization could be realized by UV light irradiation.

Highlights in Organic Chemistry [Recent Advances in the Field of Peptide Nucleic Acids

Since their development, peptide nucleic acids (PNAs) have attracted much attention as potential pharmacological regulators of gene expression. Currently, applications of PNAs are restricted through deficiencies in target specificity, aqueous solubility and cellular uptake. This highlight reviews syntheses and hybridisation properties of recent PNA analogues designed to address these limitations. Keywords: hybridisation properties, nucleobase modifications, side chain modifications, backbone modifications, PNA analogues, Peptide nucleic acids

Promising nucleic acid analogs and mimics: characteristic features and applications of PNA, LNA, and morpholino

Nucleic acid analogs and mimics are commonly the modifications of native nucleic acids at the nucleobase, the sugar ring, or the phosphodiester backbone. Many forms of promising nucleic acid analogs and mimics are available, such as locked nucleic acids (LNAs), peptide nucleic acids (PNAs), and morpholinos. LNAs, PNAs, and morpholinos can form both duplexes and triplexes and have improved biostability. They have become a general and versatile tool for DNA and RNA recognition. LNA is a general and versatile tool for specific, high-affinity recognition of single-stranded DNA (ssDNA) and single-stranded RNA (ssRNA). LNA can be used for designing LNA oligoes for hybridization studies or as real time polymerase chain reaction probes in the form of Taqman probes. LNA also has therapeutic and diagnostic applications. PNA is another type of DNA analog with neutral charge. The extreme stability of PNA makes it an ideal candidate for the antisense and antigene application. PNA is used as probe for gene cloning, mutation detection, and in homologous recombination studies. It was also used to design transcription factor decoy molecules for target gene induction. Morpholino, another structural type, was devised to circumvent cost problems associated with DNA analogs. It has become the premier knockdown tool in developmental biology due to its cytosolic delivery in the embryos by microinjection. Thus, the nucleic acid analogs provide an advantage to design and implementation, therapies, and research assays, which were not implemented due to limitations associated with standard nucleic acids chemistry.

Peptide Nucleic Acid Conjugates: Synthesis, Properties and Applications

Artificial control of gene expression has great potential in the treatment of many human diseases, and peptide nucleic acids (PNAs) offer several potential advantages for silencing gene expression in mammalian cells. The pseudopeptide backbone of the PNA makes it resistant to enzymatic degradation, and PNAs bind complementary DNA and RNA with high affinity and specificity. PNAs are potentially leading agents for antigene and antisense therapeutics, but the application of PNAs in the in vivo setting is hampered by their poor intracellular delivery. This problem has been addressed by PNA conjugation to lipophilic moieties, peptides, and cell-specific receptor ligands. The biological activity of PNAs can also benefit from conjugation to DNA interactive compounds like intercalators and alkylators. Here we review the most interesting literature concerning PNA conjugation with small molecules, emphasizing synthetic approaches, properties and applications of the PNA conjugates.