Peptide Nucleic Acid Conjugates Research Articles

Inhibition of Japanese encephalitis virus replication by peptide nucleic acids targeting cis-acting elements on the plus- and minus-strands of viral RNA

Japanese encephalitis virus (JEV) is a major cause of acute viral encephalitis in humans. The single-stranded, plus-sense viral genome, which is used for translation and minus-strand RNA synthesis, and its complementary minus-strand viral RNA contain various sequences and RNA secondary structures conserved in flaviviruses, providing potential targets for antisense agents. Here, we investigated the antiviral effects of peptide nucleic acids (PNAs) targeting cis-acting signals at the 5′-untranslated region (UTR), 3′-UTR, and genome cyclization motifs on the plus-strand RNA, as well as the 95-nucleotide 3′-end of the minus-strand RNA, which serves as a template for plus-strand RNA synthesis by the viral RNA-dependent RNA polymerase (RdRp). Among the tested cell-penetrating peptide (CPP)–PNA conjugates, a 17-mer PNA conjugate targeting the top of the 3′-UTR loop structure was most effective in suppressing virus proliferation. In vitro RdRp assays and electrophoretic mobility shift assays using a functional recombinant JEV RdRp showed that the 3′-terminal region-targeting PNAs could inhibit RNA synthesis by competing with viral RdRp for binding to a selected cis-acting element at the 3′-end of plus- and minus-strand viral RNAs. Collectively, our results suggest that CPP–PNA conjugates can suppress JEV proliferation by blocking RNA–protein or RNA–RNA interactions essential for productive viral infection.

Binding of HIV-1 TAR mRNA to a peptide nucleic acid oligomer and its conjugates with metal-ion-binding multidentate ligands

A peptide nucleic acid (PNA) oligomer and a series of PNA conjugates featuring covalently attached pendant 1,4,7,10-tetraazacyclododecane (cyclen) or bis((pyridin-2-yl)methyl)amine (DPA) moieties have been synthesized that are complementary to regions of the HIV-1 TAR messenger RNA stem-loop. Thermal denaturation studies, in conjunction win with native gel shift assays, suggest that the PNAs "invade" TAR to produce a mixture of two 1:1 PNA-TAR adducts, tentatively assigned as an "open-duplex" structure, in which the TAR stem-loop dissociates and the PNA hybridizes with its RNA complement via Watson-Crick base-pairing, and a triplex-type structure, in which the initially displaced RNA segment is bound to the PNA:RNA duplex through Hoogsteen base-pairing. Thermal denaturation experiments with the TAR sequence and single-stranded RNA and DNA oligonucleotides, both in the presence and in the absence of Zn(2+) ions, show that the introduction of cyclen or DPA ligand arms into the PNA oligomer leads to a small but reproducible increase in the T (m) values. This is attributed to hydrogen-bonding and/or electrostatic interactions between protonated forms of cyclen/DPA and the cognate RNA or DNA oligonucleotide targets. Contrary to expectations, the addition of Zn(2+) ions did not further enhance duplex formation through binding of Zn(II)-cyclen or Zn(II)-DPA moieties to the complementary RNA or DNA. Native gel shift assays further confirmed the stability increase of the metal-free cyclen- and DPA-modified PNA hybrids as compared with a control PNA sequence.

Improved cell-penetrating peptide–PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle

Steric blocking peptide nucleic acid (PNA) oligonucleotides have been used increasingly for redirecting RNA splicing particularly in therapeutic applications such as Duchenne muscular dystrophy (DMD). Covalent attachment of a cell-penetrating peptide helps to improve cell delivery of PNA. We have used a HeLa pLuc705 cell splicing redirection assay to develop a series of PNA internalization peptides (Pip) conjugated to an 18-mer PNA705 model oligonucleotide with higher activity compared to a PNA705 conjugate with a leading cell-penetrating peptide being developed for therapeutic use, (R-Ahx-R)4. We show that Pip–PNA705 conjugates are internalized in HeLa cells by an energy-dependent mechanism and that the predominant pathway of cell uptake of biologically active conjugate seems to be via clathrin-dependent endocytosis. In a mouse model of DMD, serum-stabilized Pip2a or Pip2b peptides conjugated to a 20-mer PNA (PNADMD) targeting the exon 23 mutation in the dystrophin gene showed strong exon-skipping activity in differentiated mdx mouse myotubes in culture in the absence of an added transfection agent at concentrations where naked PNADMD was inactive. Injection of Pip2a-PNADMD or Pip2b-PNADMD into the tibealis anterior muscles of mdx mice resulted in ∼3-fold higher numbers of dystrophin-positive fibres compared to naked PNADMD or (R-Ahx-R)4-PNADMD.

Effective Exon Skipping and Restoration of Dystrophin Expression by Peptide Nucleic Acid Antisense Oligonucleotides in mdx Mice

Duchenne muscular dystrophy (DMD) is the most common and severe form of muscular dystrophy, arising from mutations in the dystrophin gene that preclude the synthesis of functional protein. Antisense oligonucleotides (AOs) have been shown to induce specific exon skipping and thereby restore the reading frame and expression of functional dystrophin. In this report, we examine the effects of peptide nucleic acid (PNA) oligonucleotides and PNAs conjugated with peptides including TAT, muscle-specific peptide (MSP), adeno-associated virus 6 (AAV6) functional domain (AAV6), and AAV8 functional domain (AAV8), on exon skipping in vitro and in vivo. Efficient skipping of targeted exon 23 was achieved in cultured mdx myoblasts with PNA and PNA-peptide conjugates. Furthermore, single intramuscular injections of PNA and all PNA-peptide conjugates resulted in significant numbers of dystrophin-positive fibers in the injected tibialis anterior (TA) muscles of mdx mice, with no apparent local toxicity. Similar effects of exon skipping and dystrophin expression were obtained in mice of all ages. PNA and PNA-AAV6, PNA-AAV8 conjugates induced dystrophin expression in a dose-dependent manner. Our results demonstrate that PNAs have a higher efficiency of exon skipping than 2'O methyl phosphorothioate AOs do, and have a potential use in AO chemistry for antisense therapy of DMD.

Photochemically enhanced delivery of a cell‐penetrating peptide nucleic acid conjugate targeting human telomerase reverse transcriptase: effects on telomere status and proliferative potential of human prostate cancer cells

Peptide nucleic acids (PNAs) are DNA mimics that have been demonstrated to be efficient antisense/antigene tools in cell-free systems. However, their potential as in vivo regulators of gene expression has been hampered by their poor uptake by living cells, and strategies need to be developed for their intracellular delivery. This study has aimed to demonstrate the possibility (i) of efficiently delivering a PNA, which targets mRNA of the catalytic component of human telomerase reverse transcriptase (hTERT), into DU145 prostate cancer cells through a combined approach based on conjugation of the PNA to Tat internalizing peptide (hTERT-PNA-Tat) and subsequent photochemical internalization, and (ii) to interfere with telomerase function. Treated cells were analysed for telomerase activity, hTERT expression, growth rate, ability to undergo apoptosis and telomere status. After exposure to light, DU145 cells treated with hTERT-PNA-Tat and the photosensitiser TPPS2a showed dose-dependent inhibition of telomerase activity, which was accompanied by marked reduction of hTERT protein expression. A dose-dependent decline in DU145 cell population growth and induction of caspase-dependent apoptosis were also observed from 48 h after treatment. Such an antiproliferative effect was associated with the presence of telomeric dysfunction, as revealed by cytogenetic analysis, in the absence of telomere shrinkage, and with induction of DNA damage response as suggested by the increased expression of gamma-H2AX. Our results (i) indicate photochemical internalization as an efficient approach for intracellular delivery of chimaeric PNAs, and (ii) corroborate earlier evidence suggesting pro-survival and anti-apoptotic roles of hTERT, which are independent of its ability to maintain telomere length.

A Novel Cell-penetrating Peptide, M918, for Efficient Delivery of Proteins and Peptide Nucleic Acids

Cell-penetrating peptides (CPPs) have attracted increasing attention in the past decade as a result of their high potential to convey various, otherwise impermeable, bioactive agents across cellular plasma membranes. Albeit different CPPs have proven potent in delivery of different cargoes, there is generally a correlation between high efficacy and cytotoxicity for these peptides. Hence, it is of great importance to find new, non-toxic CPPs with more widespread delivery properties. We present a novel CPP, M918, that efficiently translocates various cells in a non-toxic fashion. In line with most other CPPs, the peptide is internalized mainly via endocytosis, and in particular macropinocytosis, but independent of glycosaminoglycans on the cell surface. In addition, in a splice correction assay using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge to M918 (M918-PNA), we observed a dose-dependent increase in correct splicing, exceeding the effect of other CPPs. Our data demonstrate that M918 is a novel CPP that can be used to translocate different cargoes inside various cells efficiently.

Label- and marker-free gene detection based on hybridization-induced conformational flexibility changes in a ferrocene–PNA conjugate probe

We report a strategy for label-free and marker-free gene detection transducing the hybridization event to an electrochemical signal based on the hybridization-induced conformational flexibility change in probe structure. The probe structure was designed to possess a ferrocene moiety as a reporter part and a cysteine moiety as an anchor part at each end of a peptide nucleic acid (PNA) as a recognition part. Electrochemical examination of probe-modified gold electrodes revealed that the ferrocene moiety was placed at the flexible end of the linear probe chain. Upon hybridization with a complementary target DNA, the resultant rigid duplex restricted the ferrocene motion to the electrode surface, causing a decrease in the observed current. The target DNA was detected with the detection limit of 1.44 x 10(-11) M. Thus the probe functioned as a 'self-reporting probe' and detection of the target DNA was demonstrated without the need for external indicators. Moreover, the sensor electrode was able repeatedly to detect the target DNA by the process of regeneration and could discriminate a mismatched DNA.

Electrospray ionisation-cleavable tandem nucleic acid mass tag–peptide nucleic acid conjugates: synthesis and applications to quantitative genomic analysis using electrospray ionisation-MS/MS

The synthesis and characterization of isotopomer tandem nucleic acid mass tag–peptide nucleic acid (TNT–PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

RNA targeting with peptide conjugates of oligonucleotides, siRNA and PNA

Towards the development of oligonucleotide analogues and siRNA as drugs, one potential alternative to the use of liposomal transfection agents is the covalent conjugation of a cell-penetrating peptide (CPP), with the intention of imparting on the oligonucleotide or siRNA an enhanced ability to enter mammalian cells and reach the appropriate RNA target. We have developed robust methods for the chemical synthesis of disulfide-linked conjugates of oligonucleotide analogues, siRNA and peptide nucleic acids (PNA) with a range of cationic and other CPPs. In a HeLa cell assay with integrated plasmid reporters of Tat-dependent trans-activation at the TAR RNA target in the cell nucleus, we were unable to obtain steric block inhibition of gene expression for conjugates of CPPs with a 12-mer oligonucleotide mixmer of 2′- O-methyl and locked nucleic acids units. By contrast, we were able to obtain some reductions in expression of P38α MAP kinase mRNA in HeLa cells using μM concentrations of Penetratin or Tat peptides conjugated to the 3′-end of the sense strand of siRNA. However, the most promising results to date have been with a 16-mer PNA conjugated to the CPP Transportan or a double CPP R 6-Penetratin, where we have demonstrated Tat-dependent trans-activation inhibition in HeLa cells. Results to date suggest the possibility of development of CPP–PNA conjugates as anti-HIV agents as well as other potential applications involving nuclear cell delivery, such as the redirection of splicing.

A Bioinformatics Approach to Ribosomal Antibiotics Research Using Peptide-Peptide Nucleic Acid (PNA) Conjugates

1* Abstract Peptide - Peptide Nucleic Acid (PNA) conjugates are known for their antibacterial effects on several pathogens. Here we designed five PNA-peptide conjugates that consist of sequences originating from conservative sequences of 16S ribosomal RNA (rRNA) from hundreds of different bacterial species. Their antibacterial effects were tested on E. coli K12 and Klebsiella pneumoniae. The result shows the sequence-dependent antibacterial or bactericidal effects of the designed peptide-PNA conjugates. We also developed a bioinformatics tool and examined any possible off-target effects of the peptide-PNA we designed. Using this tool, we also predicted the least off-target sequences for further studies. Designing peptide-PNAs utilizing conservative 16S rRNA sequences may pave a way to developing broad-spectrum ribosomal antibiotics fighting life-threatening, drug-resistant infections.

Sequence-Specific Nucleic Acid Damage Induced by Peptide Nucleic Acid Conjugates That Can Be Enzyme-Activated

Mitarbeiter gesucht: Ein Konjugat aus einer 12meren Peptidnucleinsäure (PNA) und Flavin wird mit der zur PNA-Einheit komplementären Sequenz in einem 25meren Oligo-2′-desoxyribonucleotid hybridisiert, und nun kann die Flavineinheit eine NADPH:Flavin-Oxidoreduktase „anwerben“. Mithilfe dieses Komplexes kann molekularer Sauerstoff in Gegenwart von NADH aktiviert und das Oligonucleotid-Zielmolekül selektiv oxidiert werden (siehe Schema).

Sequence‐Specific Nucleic Acid Damage Induced by Peptide Nucleic Acid Conjugates That Can Be Enzyme‐Activated

Recruitment drive: A 12 mer peptide nucleic acid(PNA)–flavin conjugate is prepared. When the PNA moiety is hybridized to its complementary sequence in a 25 mer oligo-2′-deoxyribonucleotide, the flavin moiety can recruit an NADPH:flavin oxidoreductase. This complex allows the activation of molecular oxygen in the presence of NADH and the selective oxidation of the oligonucleotide target (see scheme).

Peptide Nucleic Acid Conjugates: Synthesis, Properties and Applications

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Enhanced delivery of cell-penetrating peptide–peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.

Evaluation of Basic Amphipathic Peptides for Cellular Delivery of Antisense Peptide Nucleic Acids

Cellular permeation peptides have been used successfully for the delivery of a variety of cargoes across cellular membranes, including large hydrophilic biomolecules such as proteins, oligonucleotides, or plasmid DNA. For the present work, a series of short amphipathic peptides was designed to elucidate the structural requirements for efficient and nontoxic delivery of peptide nucleic acids (PNAs). On the basis of an idealized alpha-helical structure, the helical parameters were modulated systematically to yield peptides within a certain range of hydrophobicity and amphipathicity. The corresponding PNA conjugates were synthesized and characterized in terms of secondary structure, enzymatic stability, and antisense activity. The study revealed correlations between the physicochemical and biophysical properties of the conjugates and their biological activity and led to the development of potent peptide vectors for the cellular delivery of antisense PNAs. Two representative compounds were radiolabeled and evaluated for their biodistribution in healthy mice.

Covalent functionalization of single walled carbon nanotubes with peptide nucleic acid: Nanocomponents for molecular level electronics

Imparting molecular recognition to carbon nanotubes (CNTs) by conjugating them with bio-molecules has been an area of great interest as the resulting highly functionalized CNT-bioconjugates find their applications in various fields like molecular level electronics, pharmaceuticals, drug delivery, novel materials and many others. In this work we demonstrate the synthesis of functionally engineered single walled carbon nanotubes (SWNTs)-peptide nucleic acid (PNA) conjugates especially for nanoelectronic applications. Here we exploited the exceptional structural and chemical advantages of PNA (an artificial analogue of DNA) to join SWNTs ropes. SWNT–PNA–SWNT conjugates were synthesized using carbodiimide coupling chemistry and characterized by host of techniques like scanning electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy. The results from different techniques confirm the formation of these conjugates. Theoretical analysis of molecular orbitals obtained by quantum mechanical simulations show that the highest occupied molecular orbital is located on the glutamate linker and that this interface state will align closely to the valence band of the extended SWNT facilitating charge transfer. The unique electrical and structural properties of these conjugates make them a potential candidate for application in CNT based nanodevices.

Disulfide Conjugation of Peptides to Oligonucleotides and Their Analogs

Peptide conjugation of oligonucleotides and their analogs is being studied widely towards improving the delivery of oligonucleotides into cells. Amongst the many possible routes of conjugation, the disulfide linkage has proved to be the most popular. This reversible linkage may have advantages for cell delivery, since it is likely to be cleaved within cells, thus releasing the oligonucleotide cargo. It is straightforward to introduce thiol functionalities into both oligonucleotide and peptide components suitable for disulfide conjugation. However, severe difficulties have been encountered in carrying out conjugations between highly cationic peptides and negatively charged oligonucleotides because of aggregation and precipitation. Presented here are reliable protocols for disulfide conjugation that have been verified for both cationic and hydrophobic peptides as well as oligonucleotides containing deoxyribonucleosides, ribonucleosides, 2'-O-methylribonucleosides, locked nucleic acid (LNA) units, as well as phosphorothioate backbones. Also presented are reliable protocols for disulfide conjugation of peptide nucleic acids (PNAs) with peptides.

Evaluation of Transfection Protocols for Unmodified and Modified Peptide Nucleic Acid (PNA) Oligomers

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.

Zn2+ dependent DNA binders based on terminally modified peptide nucleic acids

Two ligand–intercalator–peptide nucleic acid conjugates (L-NADI-PNAs) have been synthesized. Affinity of these conjugates to their complementary DNAs was found to be affected by Zn2+. The magnitude of this effect could be controlled by a variation of the ligand. Upon binding Zn2+ the L-NADI-PNAs form positively charged ZnL complexes, which interact with the negatively charged DNA backbone. This electrostatic interaction stabilizes PNA/DNA duplexes. It has been found that Zn2+ dependent stabilization takes place only if the ZnL complex has a higher total positive charge than the ligand. Linear correlation has been observed between Zn2+ induced stabilization of PNA/DNA duplexes and difference of charges of the ZnL complex and the ligand.

PNA−Nitrogen Mustard Conjugates Are Effective Suppressors of HER-2/neu and Biological Tools for Recognition of PNA/DNA Interactions

Peptide nucleic acids (PNAs) are promising tools for gene regulation. One of the challenges of using PNAs as gene regulators is the need to optimize the efficiency of interaction with critical sequences of DNA. To improve the efficiency of binding between PNAs and the HER-2/neu promoter, mono- and bis-pyrimidine-rich PNAs were conjugated to a nitrogen mustard at either the amino or carboxy terminus. Gel shift analysis demonstrated that conjugation to an alkylating agent slowed PNA binding and favored PNA:DNA:DNA triplex helix formation while preserving a high binding affinity. Sites of DNA alkylation were visualized by piperidine cleavage and showed PNA binding first by Hoogsteen bond formation with the target duplex to form a stable PNA:DNA:DNA triplex structure which is later converted to a PNA:DNA:PNA triple helix by strand invasion and Watson-Crick base pairing by a second PNA molecule. In this way, PNA-directed DNA alkylation was used to deduce the mode of PNA binding. Transient transfection experiments demonstrated that the PNA-nitrogen mustard conjugates suppressed HER-2/neu expression by up to 80%. In comparison with an unmodified mono-PNA or a bis-PNA, these results indicate that the covalent adducts stabilized PNA binding in cells and suggest that the conjugation of PNAs to nitrogen mustards is a robust strategy for developing antigene PNA oligonucleotides to prevent transcription.