Peptide Nucleic Acid Oligomers Research Articles

An Amphipathic trans-Acting Phosphorothioate DNA Element Delivers Uncharged PNA and PMO Nucleic Acid Sequences in Mammalian Cells.

An innovative approach to the delivery of uncharged peptide nucleic acids (PNAs) and phosphorodiamidate morpholino (PMO) oligomers in mammalian cells is described and consists of extending the sequence of those oligomers with a short PNA-polyA or PMO-polyA tail. Recognition of the polyA-tailed PNA or PMO oligomers by an amphipathic trans-acting polythymidylic thiophosphate triester element (dTtaPS) results in efficient internalization of those oligomers in several cell lines. The authors' findings indicate that cellular uptake of the oligomers occurs through an energy-dependent mechanism and macropinocytosis appears to be the predominant endocytic pathway used for internalization. The functionality of the internalized oligomers is demonstrated by alternate splicing of the pre-mRNA encoding luciferase in HeLa pLuc 705 cells. Amphipathic phosphorothioate DNA elements may represent a unique class of cellular transporters for robust delivery of uncharged nucleic acid sequences in live mammalian cells. © 2016 by John Wiley & Sons, Inc.

Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis

Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time.

Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis.

BackgroundThermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization.ResultsHere, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe.ConclusionsThe PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-015-0027-5) contains supplementary material, which is available to authorized users.

PNA Molecular Beacons Assembled by Post-Synthetic Click Chemistry Functionalization.

To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post-synthetic on-resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide-containing monomers into the oligomer by automatic solid-phase peptide synthesis and subsequent derivatization with pyrene moieties by copper-catalyzed azide-alkyne cycloaddition. Two pyrene-based quencher-free PNA molecular beacons, a stemless MB and one possessing a stem-loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem-loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.

Incorporation of Naked Peptide Nucleic Acids into Liposomes Leads to Fast and Efficient Delivery.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.

Self-Assembled Hybrid Aptamer-Fc Conjugates for Targeted Delivery: A Modular Chemoenzymatic Approach.

Over the past decade, DNA and RNA aptamers have attracted keen research interest due to their ability to specifically bind targets of therapeutic relevance. However, their application is often hampered by a short serum half-life and missing effector functions. Conjugation of aptamers to antibody Fc fragments could improve pharmacokinetics, enable immune effector mechanisms, and provide an option for the introduction of desired payloads (e.g., toxins or fluorescent dyes). We developed a modular scaffold-supported system based on human IgG1 Fc fragments, which allows for its dual functionalization with moieties of interest. In our approach, two bioorthogonal, enzyme-mediated reactions were used in combination with oxime ligation and self-assembly based on PNA-DNA base pairing. Thus, an engineered synthetic peptide nucleic acid (PNA) oligomer was coupled to the C-termini of the Fc dimer upon sequence-specific sortase A-mediated transpeptidation. Hybridization of the resulting Fc-PNA conjugate with a tailored DNA aptamer that binds cancer-related hepatocyte growth factor receptor (c-MET) led to a hybrid construct which showed strong and specific binding to c-MET and was readily internalized by c-MET-overexpressing cells. To install an additional orthogonally addressable site, aldehyde tag technology was applied followed by oxime ligation with an aminooxy-bearing fluorescent dye as model cargo. Delivery of fluorescent probe specifically to c-MET-overexpressing cells was confirmed by flow cytometry. Our approach can provide access to engineered aptamer-Fc conjugates with desired target specificity and cytotoxic payloads.

Application of fluorescence melting curve analysis for dual DNA detection using single peptide nucleic acid probe.

Peptide nucleic acid (PNA) is an artificially synthesized polymer. PNA oligomers show greater specificity in binding to complementary DNAs. Using this PNA, fluorescence melting curve analysis (FMCA) for dual detection was established. Genomic DNA of Mycoplasma fermentans and Mycoplasma hyorhinis was used as a template DNA model. By using one PNA probe, M. fermentans and M. hyorhinis could be detected and distinguished simultaneously in a single tube. The developed PNA probe is a dual-labeled probe with fluorescence and quencher dye. The PNA probe perfectly matches the M. fermentans 16s rRNA gene, with a melting temperature of 72°C. On the other hand, the developed PNA probe resulted in a mismatch with the 16s rRNA gene of M. hyorhinis, with a melting temperature of 44-45°C. The melting temperature of M. hyorhinis was 27-28°C lower than that of M. fermentans. Due to PNA's high specificity, this larger melting temperature gap is easy to create. FMCA using PNA offers an alternative method for specific DNA detection.

Synthesis of anionic peptide nucleic acid oligomers including γ-carboxyethyl thymine monomers

Synthesis of a modified thymine monomer based on l-Glu, solid phase synthesis of anionic peptide nucleic acid (PNA) oligomers containing the modified monomer and optimization of the deblocking procedure and cleavage conditions of the PNA oligomers from resin are accomplished.

Electroporation Enhanced Effect of Dystrophin Splice Switching PNA Oligomers in Normal and Dystrophic Muscle.

Peptide nucleic acid (PNA) is a synthetic DNA mimic that has shown potential for discovery of novel splice switching antisense drugs. However, in vivo cellular delivery has been a limiting factor for development, and only few successful studies have been reported. As a possible modality for improvement of in vivo cellular availability, we have investigated the effect of electrotransfer upon intramuscular (i.m.) PNA administration in vivo. Antisense PNA targeting exon 23 of the murine dystrophin gene was administered by i.m. injection to the tibialis anterior (TA) muscle of normal NMRI and dystrophic mdx mice with or without electroporation. At low, single PNA doses (1.5, 3, or 10 µg/TA), electroporation augmented the antisense exon skipping induced by an unmodified PNA by twofold to fourfold in healthy mouse muscle with optimized electric parameters, measured after 7 days. The PNA splice switching was detected at the RNA level up to 4 weeks after a single-dose treatment. In dystrophic muscles of the MDX mouse, electroporation increased the number of dystrophin-positive fibers about 2.5-fold at 2 weeks after a single PNA administration compared to injection only. In conclusion, we find that electroporation can enhance PNA antisense effects in muscle tissue.

Abstract 2926: PNA as novel targeted cancer therapy for BRAFV600E mutant melanoma

Abstract Our aim is to target tumor cells specifically by directly suppressing their oncogenes with peptide nucleic acid (PNA) oligonucleotide analogues. PNA oligonucleotides bind to DNA over 1000-fold more avidly than its native complement, are completely resistant to intra and extra cellular enzymatic degradation, show no nonspecific toxicity at therapeutic levels, and when conjugated to delivery peptides can be made nuclear and cell membrane permeable. We have employed these PNA oligomers to target BRAFV600E which is prevalent in cutaneous melanoma in a sequence-specific complementary manner so as to disrupt transcription by strand invasion. For these studies, we have developed a novel delivery peptide conjugated to PNA modified to increase both cellular delivery and PNA stability towards its target. Our results indicate that exposure of the melanoma cell lines to a modified PNA-peptide conjugate selective for BRAFV600E results in a concentration-dependent inhibition of cell growth that is specific for the BRAFV600E mutant melanoma cell lines with an IC50 range of 250 to 500 nM. Moreover, there is no inhibition of BRAFWT cell growth at these concentrations. This is associated with suppression of BRAFV600E protein over time with no effect on BRAFWT protein levels. Furthermore, BRAFV600E protein expression was suppressed for up to 6 days following initial exposure proving the durability of this type of inhibition. Exposure to this modified PNA-peptide down-regulates BRAFV600E mRNA transcription exclusively in the mutant cell lines. Live cell imaging of BRAFV600E mutant cells confirms localization of fluorescein-labeled PNA-delivery peptide specific to BRAFV600E to the nucleus within 3 hours of treatment. Our results indicate that these PNA-peptide derivatives could represent a novel and promising new therapy for patients with BRAFV600E mutant melanoma, and this technology could be applied to a multitude of other cancers either with specific translocations or mutations differing from wild-type cells even by only a single base pair. Citation Format: Jeffrey H. Rothman, Oliver Surriga, Shyamprasad D. Vasudeva, Grazia Ambrosini, Ouathek Ouerfelli, Gary K. Schwartz. PNA as novel targeted cancer therapy for BRAFV600E mutant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2926. doi:10.1158/1538-7445.AM2014-2926

Using triple-helix-forming Peptide nucleic acids for sequence-selective recognition of double-stranded RNA.

Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple-helix-forming peptide nucleic acids (PNAs) that bind in the major grove of the RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M), which enables strong triple-helical binding under physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E), which enable recognition of isolated pyrimidines in the purine-rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis, HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included.

Conformational studies of chiral d-Lys-PNA and achiral PNA system in binding with DNA or RNA through a molecular dynamics approach

The growing interest in peptide nucleic acid (PNA) oligomers has led to the development of a very wide variety of PNA derivatives. Among others, the introduction of charged chiral groups on a PNA oligomer has proven effective in improving DNA binding ability, complexation direction and cellular uptake. In particular, the introduction of three adjacent chiral monomers based on D-Lys in the middle of the PNA sequence (D-Lys-PNA) has produced noteworthy results in modulating the directionality of the binding with the DNA complementary strand and in mismatch detection. Here, through a molecular dynamics approach, a comparative study has been carried out to investigate the structural properties that drive the interaction of the chiral D-Lys-PNA and the corresponding achiral PNA system with DNA as well as RNA complementary strands, starting from the crystal structure of D-Lys-PNA in complex with DNA. The results obtained complement experimental data and indicate that the binding with the RNA molecule, compared to DNA, is differently affected by the addition of three D-Lys groups on the PNA backbone, suggesting that this modification could be taken into account for the development of new PNA-based molecules able to discriminate between DNA and RNA.

Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 μM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.

Preparation and evaluation of a peptide nucleic acid gene chip system associated with surface plasmon resonance

The aim of this study was to construct a gene chip system based on a surface plasmon resonance technique, where peptide nucleic acid (PNA) oligomers are used as probes. Since the self-assembled monolayer (SAM) technology offers good control at the molecular level, we prepared 2D surface chemistry via SAM for probe attachments. PNA, which was designed according to the bioinformatics, was immobilized on the SAM-modified chip, and subsequently, relevant parameters of the experiment were ensured and optimized. Our results suggest that the ion strength and pH value of the buffer solution do not play significant roles in PNA or its complementary strand hybridization. The PNA probe binds to its complementary nucleic acid strand with a higher sensitivity and specificity compared to those of a traditional DNA probe. The PNA probe combined with surface plasmon resonance (SPR) technology has the benefits of being a label-free and in-real time monitor, as well as having improved hybridization and stability efficiency, which highlight the PNA gene chip detection system as a promising biosensor for clinical applications.

The Single-Molecule Conductance and Electrochemical Electron-Transfer Rate Are Related by a Power Law

This study examines quantitative correlations between molecular conductances and standard electrochemical rate constants for alkanes and peptide nucleic acid (PNA) oligomers as a function of the length, structure, and charge transport mechanism. The experimental data show a power-law relationship between conductances and charge transfer rates within a given class of molecules with the same bridge chemistry, and a lack of correlation when a more diverse group of molecules is compared, in contrast with some theoretical predictions. Surprisingly, the PNA duplexes exhibit the lowest charge-transfer rates and the highest molecular conductances. The nonlinear rate-conductance relationships for structures with the same bridging chemistries are attributed to differences in the charge-mediation characteristics of the molecular bridge, energy barrier shifts and electronic dephasing, in the two different experimental settings.

ChemInform Abstract: Ionic Liquid Mediated Synthesis of Peptide Nucleic Acids Dimers.

The unprecedented use of ionic liquids in the synthesis of peptide nucleic acids (PNA) oligomers has been investigated. As a proof of concept, the preparation of PNA dimers in ILs has been performed using last generation coupling reagents. More importantly, the application of the supported ionic liquid phase (SILP) technique to the preparation of a PNA dimer has been achieved, including loading, chain elongation and cleavage from the IL support of the PNA fragment.

Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5

The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.

Synthesis of Stable Peptide Nucleic Acid‐Modified Gold Nanoparticles and their Assembly onto Gold Surfaces

PNA does it better: Peptide nucleic acid (PNA) oligomers were attached to gold nanoparticles (AuNPs) through a variety of mono- and trithiol linkers. These functionalized particles had sufficient stability for sequence-specific self-assembly onto gold surfaces (see figure) in the absence of ions or surfactants. The nanoparticle surface densities obtained were superior to comparable DNA-modified AuNPs.

Peptide Nucleic Acids (PNAs) Patterning by an Automated Microarray Synthesis System Through Photolithography

Peptide nucleic acids (PNA) microarray assembled with hundreds of unique PNA oligomers has been regarded as a new and mighty competitor of DNA chip in gene analyzing. However, PNA microarray is still a luxury art due to the difficult and laborious chemical synthesis. Herein, we have developed a fully-automated synthesizer for PNA microarray through photolithography. A preactivation mixer was designed and integrated into the synthesizer in order to get rid of the annoying manual process and increase the coupling efficiency of PNA monomers. The PNA patterning model was carried out to check the performance of the automated synthesizer, revealing that an exposure time of 3 min was sufficient for the complete removal of o-nitroveratryloxycarbonyl (NVOC) groups from the synthetic sites with the help of photosensitizer isopropylthioxanthone and the stepwise yield was measured to be about 98.0%, which is comparable with that from conventional fluorenyl-methyloxycarbonyl (FMOC) chemistry. Those results have definitely demonstrated the possibility and capability of this fully-automated synthesizer to fabricate high-quality PNA microarrays.

Detection and discrimination of recombinant plasmid encoding hepatitis C virus core/E1 gene based on PNA and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for direct detection and discrimination of double-stranded plasmid (ds-Pl) without the need for denaturation of the target plasmid sample using a peptide nucleic acid (PNA) oligomer as the probe is described. This goal was achieved by modification of gold electrode with 6-mercapto-1-hexanol following monolayer self-assembly of cysteine conjugated 20-mer PNA oligomer probe, complementary to the HCV core/E1 region, which binds to ds-Pl and forms PNA/ds-Pl structure. The significant variation in differential pulse voltammetric response of methylene blue on the probe modified electrode upon contacting with complementary double-strand plasmid to form PNA/ds-Pl triplex structure is the principle of target plasmid detection. The results indicated that the reduction peak current was linear with the concentration of complementary strand in the range of 10–300pg/μl with a detecion limit of 9.5pg/μl.