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1.  Base pair opening kinetics study of the aegPNA:DNA hydrid duplex containing a site-specific GNA-like chiral PNA monomer 
Nucleic Acids Research  2011;39(16):7329-7335.
Peptide nucleic acids (PNA) are one of the most widely used synthetic DNA mimics where the four bases are attached to a N-(2-aminoethyl)glycine (aeg) backbone instead of the negative-charged phosphate backbone in DNA. We have developed a chimeric PNA (chiPNA), in which a chiral GNA-like γ3T monomer is incorporated into aegPNA backbone. The base pair opening kinetics of the aegPNA:DNA and chiPNA:DNA hybrid duplexes were studied by NMR hydrogen exchange experiments. This study revealed that the aegPNA:DNA hybrid is much more stable duplex and is less dynamic compared to DNA duplex, meaning that base pairs are opened and reclosed much more slowly. The site-specific incorporation of γ3T monomer in the aegPNA:DNA hybrid can destabilize a specific base pair and its neighbors, maintaining the thermal stabilities and dynamic properties of other base pairs. Our hydrogen exchange study firstly revealed the unique kinetic features of base pairs in the aegPNA:DNA and chiPNA:DNA hybrids, which will provide an insight into the development of methodology for specific DNA recognition using PNA fragments.
PMCID: PMC3167616  PMID: 21586589
2.  Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues. 
Nucleic Acids Research  1998;26(2):566-575.
In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.
PMCID: PMC147292  PMID: 9421517
3.  Incorporation of thio-pseudoisocytosine into triplex-forming peptide nucleic acids for enhanced recognition of RNA duplexes 
Nucleic Acids Research  2014;42(6):4008-4018.
Peptide nucleic acids (PNAs) have been developed for applications in biotechnology and therapeutics. There is great potential in the development of chemically modified PNAs or other triplex-forming ligands that selectively bind to RNA duplexes, but not single-stranded regions, at near-physiological conditions. Here, we report on a convenient synthesis route to a modified PNA monomer, thio-pseudoisocytosine (L), and binding studies of PNAs incorporating the monomer L. Thermal melting and gel electrophoresis studies reveal that L-incorporated 8-mer PNAs have superior affinity and specificity in recognizing the duplex region of a model RNA hairpin to form a pyrimidine motif major-groove RNA2–PNA triplex, without appreciable binding to single-stranded regions to form an RNA–PNA duplex or, via strand invasion, forming an RNA–PNA2 triplex at near-physiological buffer condition. In addition, an L-incorporated 8-mer PNA shows essentially no binding to single-stranded or double-stranded DNA. Furthermore, an L-modified 6-mer PNA, but not pseudoisocytosine (J) modified or unmodified PNA, binds to the HIV-1 programmed −1 ribosomal frameshift stimulatory RNA hairpin at near-physiological buffer conditions. The stabilization of an RNA2–PNA triplex by L modification is facilitated by enhanced van der Waals contacts, base stacking, hydrogen bonding and reduced dehydration energy. The destabilization of RNA–PNA and DNA–PNA duplexes by L modification is due to the steric clash and loss of two hydrogen bonds in a Watson–Crick-like G–L pair. An RNA2–PNA triplex is significantly more stable than a DNA2–PNA triplex, probably because the RNA duplex major groove provides geometry compatibility and favorable backbone–backbone interactions with PNA. Thus, L-modified triplex-forming PNAs may be utilized for sequence-specifically targeting duplex regions in RNAs for biological and therapeutic applications.
PMCID: PMC3973316  PMID: 24423869
4.  Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells 
Nucleic Acids Research  2008;36(13):4424-4432.
In the search of facile and efficient methods for cellular delivery of peptide nucleic acids (PNA), we have synthesized PNAs conjugated to oligophosphonates via phosphonate glutamine and bis-phosphonate lysine amino acid derivatives thereby introducing up to twelve phosphonate moieties into a PNA oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero-duplex. These results indicate that conjugation of phosphonate moieties to the PNA can dramatically improve cellular delivery mediated by cationic lipids without affecting on the binding affinity and sequence discrimination ability, exhibiting EC50 values down to one nanomolar. Thus the intracellular efficacy of PNA oligomers rival that of siRNA and the results therefore emphasize that provided sufficient in vivo bioavailability of PNA can be achieved these molecules may be developed into potent gene therapeutic drugs.
PMCID: PMC2490735  PMID: 18596083
5.  A peptide nucleic acid (PNA) is more rapidly internalized in cultured neurons when coupled to a retro-inverso delivery peptide. The antisense activity depresses the target mRNA and protein in magnocellular oxytocin neurons. 
Nucleic Acids Research  1998;26(21):4910-4916.
A peptide nucleic acid (PNA) antisense for the AUG translation initiation region of prepro-oxytocin mRNA was synthesized and coupled to a r etro-inverso peptide that is rapidly taken up by cells. This bioconjugate was internalized by cultured cerebral cortex neurons within minutes, according to the specific property of the vector peptide. The PNA alone also entered the cells, but more slowly. Cell viability was unaffected when the PNA concentrations were lower than 10 microM and incubation times less than for 24 h. Magnocellular neurons from the hypothalamic supraoptic nucleus, which produce oxytocin and vasopressin, were cultured in chemically defined medium. Both PNA and vector peptide-PNA depressed the amounts of the mRNA coding for prepro-oxytocin in these neurons. A scrambled PNA had no effect and the very cognate prepro-vasopressin mRNA was not affected. The antisense PNA also depressed the immunocytochemical signal for prepro-oxytocin in this culture in a dose- and time-dependent manner. These results show that PNAs driven by the retro-inverso vector peptide are powerful antisense reagents for use on cells in culture.
PMCID: PMC147921  PMID: 9776752
6.  Antiproliferative effect in chronic myeloid leukaemia cells by antisense peptide nucleic acids 
Nucleic Acids Research  2002;30(17):3712-3721.
Peptide nucleic acid (PNA) is a synthetic DNA analogue that is resistant to nucleases and proteases and binds with exceptional affinity to RNA. Because of these properties PNA has the potential to become a powerful therapeutic agent to be used in vivo. Until now, however, the use of PNA in vivo has not been much investigated. Here, we have attempted to reduce the expression of the bcr/abl oncogene in chronic myeloid leukaemia KYO-1 cells using a 13mer PNA sequence (asPNA) designed to hybridise to the b2a2 junction of bcr/abl mRNA. To enhance cellular uptake asPNA was covalently linked to the basic peptide VKRKKKP (NLS-asPNA). Moreover, to investigate the cellular uptake by confocal microscopy, both PNAs were linked by their N-terminus to fluorescein (FL). Studies of uptake, carried out at 4 and 37°C on living KYO-1 cells stained with hexidium iodide, showed that both NLS-asPNA-FL and asPNA-FL were taken up by the cells, through a receptor-independent mechanism. The intracellular amount of NLS-asPNA-FL was about two to three times higher than that of asPNA-FL. Using a semi-quantitative RT– PCR technique we found that 10 µM asPNA and NLS-asPNA reduced the level of b2a2 mRNA in KYO-1 cells to 20 ± 5% and 60 ± 10% of the control, respectively. Western blot analysis showed that asPNA promoted a significant inhibition of p210BCR/ABL protein: residual protein measured in cells exposed for 48 h to asPNA was ∼35% of the control. Additionally, asPNA impaired cell growth to 50 ± 5% of the control and inhibited completion of the cell cycle. In summary, these results demonstrate that a PNA 13mer is taken up by KYO-1 cells and is capable of producing a significant and specific down-regulation of the bcr/abl oncogene involved in leukaemogenesis.
PMCID: PMC137404  PMID: 12202756
7.  Additive antisense effects of different PNAs on the in vitro translation of the PML/RARalpha gene. 
Nucleic Acids Research  1998;26(8):1934-1938.
The potential use of peptide nucleic acid (PNA) as a sequence-specific inhibitor of RNA translation is investigated in this report. Three different regions of the PML/RARalpha oncogene, including two AUG potential start codons, were studied as targets of translation inhibition by antisense PNA in a cell-free system. A PNA targeted to the second AUG start codon, which was shown previously to be able to suppress in vitro translation from that site completely, was used alone or in combination with another PNA directed to the first AUG, and a third PNA within the 5'-untranslated region (5'-UTR) of mRNA. When used alone, no PNA was able to completely block the synthesis of the PML/RARalpha protein. The 5'-UTR PNA was the most potent translation inhibitor when used as single agent. However, a near complete (>/=90%) specific inhibition of the PML/RARalpha gene was obtained when the three PNAs were used in combination, thus obtaining an additive antisense effect.
PMCID: PMC147505  PMID: 9518485
8.  DNA assembly using bis-peptide nucleic acids (bisPNAs) 
Nucleic Acids Research  2002;30(13):2782-2789.
DNA nanostructures are ordered oligonucleotide arrangements that have applications for DNA computers, crystallography, diagnostics and material sciences. Peptide nucleic acid (PNA) is a DNA/RNA mimic that offers many advantages for hybridization, but its potential for application in the field of DNA nanotechnology has yet to be thoroughly examined. We report the synthesis and characterization of tethered PNA molecules (bisPNAs) designed to assemble two individual DNA molecules through Watson–Crick base pairing. The spacer regions linking the PNAs were varied in length and contained amino acids with different electrostatic properties. We observed that bisPNAs effectively assembled oligonucleotides that were either the exact length of the PNA or that contained overhanging regions that projected outwards. In contrast, DNA assembly was much less efficient if the oligonucleotides contained overhanging regions that projected inwards. Surprisingly, the length of the spacer region between the PNA sequences did not greatly affect the efficiency of DNA assembly. Reasons for inefficient assembly of inward projecting DNA oligonucleotides include non-sequence-specific intramolecular interactions between the overhanging region of the bisPNA and steric conflicts that complicate simultaneous binding of two inward projecting strands. These results suggest that bisPNA molecules can be used for self-assembling DNA nanostructures provided that the arrangement of the hybridizing DNA oligonucleotides does not interfere with simultaneous hybridization to the bisPNA molecule.
PMCID: PMC117044  PMID: 12087161
9.  Antisense properties of duplex- and triplex-forming PNAs. 
Nucleic Acids Research  1996;24(3):494-500.
The potential of peptide nucleic acids (PNAs) as specific inhibitors of translation has been studied. PNAs with a mixed purine/pyrimidine sequence form duplexes, while homopyrimidine PNAs form (PNA)2/RNA triplexes with complementary sequences on RNA. We show here that neither of these PNA/RNA structures are substrates for RNase H. Translation experiments in cell-free extracts showed that a 15mer duplex-forming PNA blocked translation in a dose-dependent manner when the target was 5'-proximal to the AUG start codon on the RNA, whereas similar 10-, 15- or 20mer PNAs had no effect when targeted towards sequences in the coding region. Triplex-forming 10mer PNAs were efficient and specific antisense agents with a target overlapping the AUG start codon and caused arrest of ribosome elongation with a target positioned in the coding region of the mRNA. Furthermore, translation could be blocked with a 6mer bisPNA or with a clamp PNA, forming partly a triplex, partly a duplex, with its target sequence in the coding region of the mRNA.
PMCID: PMC145651  PMID: 8602363
10.  Inhibition of Growth and Gene Expression by PNA-peptide Conjugates in Streptococcus pyogenes 
While Streptococcus pyogenes is consistently susceptible toward penicillin, therapeutic failure of penicillin treatment has been reported repeatedly and a considerable number of patients exhibit allergic reactions to this substance. At the same time, streptococcal resistance to alternative antibiotics, e.g., macrolides, has increased. Taken together, these facts demand the development of novel therapeutic strategies. In this study, S. pyogenes growth was inhibited by application of peptide-conjugated antisense-peptide nucleic acids (PNAs) specific for the essential gyrase A gene (gyrA). Thereby, HIV-1 Tat peptide-coupled PNAs were more efficient inhibitors of streptococcal growth as compared with (KFF)3K-coupled PNAs. Peptide-anti-gyrA PNAs decreased the abundance of gyrA transcripts in S. pyogenes. Growth inhibition by antisense interference was enhanced by combination of peptide-coupled PNAs with protein-level inhibitors. Antimicrobial synergy could be detected with levofloxacin and novobiocin, targeting the gyrase enzyme, and with spectinomycin, impeding ribosomal function. The prospective application of carrier peptide-coupled antisense PNAs in S. pyogenes covers the use as an antimicrobial agent and the employment as a knock-down strategy for the investigation of virulence factor function.
PMCID: PMC3889189  PMID: 24193033
antimicrobial; antisense; cell-penetrating peptides; HIV-1 peptide; PNA; Streptococcus pyogenes
11.  Phospholipid conjugate for intracellular delivery of peptide nucleic acids 
Bioconjugate chemistry  2009;20(9):1729-1736.
Peptide nucleic acids (PNAs) have a number of attractive features that have made them an ideal choice for antisense and antigene-based tools, probes and drugs, but their poor membrane permeability has limited their application as therapeutic or diagnostic agents. Herein we report a general method for the synthesis of phospholipid-PNAs (LP-PNAs), and compare the effect of non-cleavable lipids and bioreductively cleavable lipids (L and LSS) and phospholipid (LP) on the splice-correcting bioactivity of a PNA bearing the cell penetrating Arg9 group (PNA-R9). While the three constructs show similar and increasing bioactivity at 1–3 μM, the activity of LP-PNA-R9 continues to increase from 4–6 μM while the activity of L-PNA-R9 remains constant and LSS-PNA-R9 decreases rapidly in parallel with their relative cytotoxicity. The activity of both LP-PNA-R9 and L-PNA-R9 were found to dramatically increase with chloroquine, as expected for an endocytotic entry mechanism. Both constructs were also found to have CMC values of 1.0 and 4.5 μM in 150 mM NaCl, pH 7 water, suggesting that micelle formation may play a hitherto unrecognized role in modulating toxicity and/or facilitating endocytosis.
PMCID: PMC2763590  PMID: 19678628
peptide nucleic acid; phospholipid; lipid; cell penetrating peptide; micelle; endocytosis; bioreductively cleavable
12.  Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions 
BMC Cancer  2010;10:342.
Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells.
We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT).
We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone.
We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs.
PMCID: PMC2910690  PMID: 20591158
13.  Peptide Nucleic Acid-Based Array for Detecting and Genotyping Human Papillomaviruses▿  
Journal of Clinical Microbiology  2009;47(6):1785-1790.
We describe a novel array for accurate and reliable genotyping of human papillomavirus (HPV) using peptide nucleic acid (PNA) probes. In order to exploit the superior hybridization properties of PNA with target HPV DNAs, we developed a novel PNA array (PANArray HPV). PANArray HPV enables the detection and genotyping of HPVs using 32 type-specific PNA capture probes for medically important HPVs. All tested HPV types showed highly unique hybridization patterns with type-specific PNA probes. PNA array results showed stable specificities and sensitivities after up to 13 months of storage at room temperature. Also, we demonstrated the superior specificity, sensitivity, and stability of PNA arrays for HPV genotyping. We compared the genotyping results of the PNA array to sequencing with MY09/11 PCR products derived from 72 clinical samples. The results showed excellent agreement between the PNA array and sequencing, except for samples reflecting multiple infections. The results from the PNA array were compared with those of type-specific PCR when discrepant results occurred owing to multiple infections. The results for the PNA array matched those of type-specific PCR in all cases. Newly developed PNA arrays show excellent specificity and sensitivity and long shelf life. Our results suggest that the PNA array represents a reliable alternative to conventional DNA arrays for HPV genotyping, as well as for diagnostics.
PMCID: PMC2691110  PMID: 19369432
14.  Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5 
Artificial DNA, PNA & XNA  2013;4(2):49-57.
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 (CCR5) 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.
PMCID: PMC3771998  PMID: 23954968
CCR5; PEG; PNA; antisense; nanoparticle; γPNA
15.  Effective delivery of antisense peptide nucleic acid oligomers into cells by anthrax protective antigen 
Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protective antigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant β-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)8] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)8 with but not without PA also corrected the IVS2-654 β-globin splice defect in cultured erythroid precursor cells from a patient with β-thalassemia [genotype, IVS2-654(β0/βE)], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells.
PMCID: PMC2581503  PMID: 18774771
peptide nucleic acid; antisense; anthrax protective antigen
16.  A Peptide Nucleic Acid-Aminosugar Conjugate Targeting Transactivation Response Element of HIV-1 RNA Genome Shows a High Bioavailability in Human Cells and Strongly Inhibits Tat-mediated Transactivation of HIV-1 Transcription 
Journal of Medicinal Chemistry  2012;55(13):6021-6032.
The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16 mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic condition, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment since co-treatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application.
PMCID: PMC3400927  PMID: 22698070
Peptide nucleic acids; polyamide nucleic acids; aminosugar; aminoglycoside; 6-aminoglucosamine; neosamine; neamine; antiviral activity; HIV; cellular penetration; TAR; Tat
17.  Convenient and Scalable Synthesis of Fmoc-Protected Peptide Nucleic Acid Backbone 
Journal of Nucleic Acids  2012;2012:354549.
The peptide nucleic acid backbone Fmoc-AEG-OBn has been synthesized via a scalable and cost-effective route. Ethylenediamine is mono-Boc protected, then alkylated with benzyl bromoacetate. The Boc group is removed and replaced with an Fmoc group. The synthesis was performed starting with 50 g of Boc anhydride to give 31 g of product in 32% overall yield. The Fmoc-protected PNA backbone is a key intermediate in the synthesis of nucleobase-modified PNA monomers. Thus, improved access to this molecule is anticipated to facilitate future investigations into the chemical properties and applications of nucleobase-modified PNA.
PMCID: PMC3400375  PMID: 22848796
18.  End invasion of peptide nucleic acids (PNAs) with mixed-base composition into linear DNA duplexes 
Nucleic Acids Research  2005;33(17):e146.
Peptide nucleic acid (PNA) is a synthetic DNA mimic with valuable properties and a rapidly growing scope of applications. With the exception of recently introduced pseudocomplementary PNAs, binding of common PNA oligomers to target sites located inside linear double-stranded DNAs (dsDNAs) is essentially restricted to homopurine–homopyrimidine sequence motifs, which significantly hampers some of the PNA applications. Here, we suggest an approach to bypass this limitation of common PNAs. We demonstrate that PNA with mixed composition of ordinary nucleobases is capable of sequence-specific targeting of complementary dsDNA sites if they are located at the very termini of DNA duplex. We then show that such targeting makes it possible to perform capturing of designated dsDNA fragments via the DNA-bound biotinylated PNA as well as to signal the presence of a specific dsDNA sequence, in the case a PNA beacon is employed. We also examine the PNA–DNA conjugate and prove that it can initiate the primer-extension reaction starting from the duplex DNA termini when a DNA polymerase with the strand-displacement ability is used. We thus conclude that recognition of duplex DNA by mixed-base PNAs via the end invasion has a promising potential for site-specific and sequence-unrestricted DNA manipulation and detection.
PMCID: PMC1243805  PMID: 16204449
19.  Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo 
Nucleic Acids Research  2006;34(8):2247-2257.
To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms.
PMCID: PMC1456331  PMID: 16670431
20.  Cyclopentane-Peptide Nucleic Acids for Qualitative, Quantitative, and Repetitive Detection of Nucleic Acids 
Analytical chemistry  2012;85(1):251-257.
We report the development of chemically-modified peptide nucleic acids (PNAs) as probes for qualitative and quantitative detection of DNA. The remarkable stability of PNAs toward enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device that can be used outside of a laboratory setting. Using an enzyme-linked reporter assay, we demonstrate that excellent levels of detection and accuracy for anthrax DNA can be achieved using PNA probes with suitable chemical components designed into the probe. In addition, we report on DNA-templated crosslinking of PNA probes as a way to preserve genetic information for repetitive and subsequent analysis. This report is the first detailed examination of the qualitative and quantitative properties of chemically-modified PNA for nucleic acid detection and provides a platform for studying and optimizing PNA probes prior to incorporation into new technological platforms.
PMCID: PMC3535555  PMID: 23214925
21.  Improved cell-penetrating peptide–PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle 
Nucleic Acids Research  2008;36(20):6418-6428.
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.
PMCID: PMC2582604  PMID: 18842625
22.  Recent Advances in Chemical Modification of Peptide Nucleic Acids 
Journal of Nucleic Acids  2012;2012:518162.
Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification.
PMCID: PMC3443988  PMID: 22991652
23.  Inhibition of Multidrug Resistance by SV40 Pseudovirion Delivery of an Antigene Peptide Nucleic Acid (PNA) in Cultured Cells 
PLoS ONE  2011;6(3):e17981.
Peptide nucleic acid (PNA) is known to bind with extraordinarily high affinity and sequence-specificity to complementary nucleic acid sequences and can be used to suppress gene expression. However, effective delivery into cells is a major obstacle to the development of PNA for gene therapy applications. Here, we present a novel method for the in vitro delivery of antigene PNA to cells. By using a nucleocapsid protein derived from Simian virus 40, we have been able to package PNA into pseudovirions, facilitating the delivery of the packaged PNA into cells. We demonstrate that this system can be used effectively to suppress gene expression associated with multidrug resistance in cancer cells, as shown by RT-PCR, flow cytometry, Western blotting, and cell viability under chemotherapy. The combination of PNA with the SV40-based delivery system is a method for suppressing a gene of interest that could be broadly applied to numerous targets.
PMCID: PMC3062552  PMID: 21445346
24.  Improvement of sequence selectivity in triple helical recognition of RNA by phenylalanine-derived PNA 
Artificial DNA, PNA & XNA  2013;4(3):69-76.
Modified peptide nucleic acids (PNA) containing one or two thymine PNA monomers derived from phenylalanine were synthesized. Triple helix formation by these modified PNAs with RNA and DNA hairpins having a variable base pair in the middle of the helix were studied using isothermal titration calorimetry and compared with triple helix formation by non-modified PNAs. While unmodified PNA had low sequence selectivity against mismatched hairpins, introduction of one or two phenylalanine-derived monomers significantly increased the mismatch discrimination and sequence selectivity of the modified PNA. Consistent with our previous observations, PNA formed more stable triple helices with RNA than with DNA. Interestingly, the phenylalanine modification further improved the preference of PNA for RNA over DNA hairpin.
PMCID: PMC3962516  PMID: 24104925
PNA backbone modification; PNA-RNA triple helix; isothermal titration calorimetry; peptide nucleic acid; recognition of double-stranded RNA
25.  Development of Peptide Nucleic Acid Probes for Detection of the HER2 Oncogene 
PLoS ONE  2013;8(4):e58870.
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.
PMCID: PMC3622650  PMID: 23593123

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