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1.  Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods 
Artificial DNA, PNA & XNA  2012;3(1):22-30.
Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.
doi:10.4161/adna.19906
PMCID: PMC3368813  PMID: 22679530
antisense; cellular delivery; lipoplex; octaarginine (CPP); peptide nucleic acid (PNA); polyethyleneimine (PEI)
2.  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.
doi:10.1093/nar/gkn401
PMCID: PMC2490735  PMID: 18596083
3.  Down-regulation of MDM2 and activation of p53 in human cancer cells by antisense 9-aminoacridine–PNA (peptide nucleic acid) conjugates 
Nucleic Acids Research  2004;32(16):4893-4902.
A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr–PNA) could be effectively delivered to JAR cells (as well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr–PNA conjugates, one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 μM caused a reduction of MDM2 levels to ∼20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.
doi:10.1093/nar/gkh820
PMCID: PMC519114  PMID: 15371552
4.  Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells 
Nucleic Acids Research  2005;33(21):6837-6849.
The trans-activation response (TAR) RNA stem–loop that occurs at the 5′ end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem–loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R6-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP–PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP–PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition.
doi:10.1093/nar/gki991
PMCID: PMC1301599  PMID: 16321967
5.  Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide 
Nucleic Acids Research  2007;35(13):4495-4502.
Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated.
doi:10.1093/nar/gkm418
PMCID: PMC1934994  PMID: 17584792
6.  Synthesis and Splice-Redirecting Activity of Branched, Arginine-Rich Peptide Dendrimer Conjugates of Peptide Nucleic Acid Oligonucleotides 
Bioconjugate Chemistry  2010;21(10):1902-1911.
Arginine-rich cell-penetrating peptides have found excellent utility in cell and in vivo models for enhancement of delivery of attached charge-neutral PNA or PMO oligonucleotides. We report the synthesis of dendrimeric peptides containing 2- or 4-branched arms each having one or more R-Ahx-R motifs and their disulfide conjugation to a PNA705 splice-redirecting oligonucleotide. Conjugates were assayed in a HeLa pLuc705 cell assay for luciferase up-regulation and splicing redirection. Whereas 8-Arg branched peptide−PNA conjugates showed poor activity compared to a linear (R-Ahx-R)4−PNA conjugate, 2-branched and some 4-branched 12 and 16 Arg peptide−PNA conjugates showed activity similar to that of the corresponding linear peptide−PNA conjugates. Many of the 12- and 16-Arg conjugates retained significant activity in the presence of serum. Evidence showed that biological activity in HeLa pLuc705 cells of the PNA conjugates of branched and linear (R-Ahx-R) peptides is associated with an energy-dependent uptake pathway, predominantly clathrin-dependent, but also with some caveolae dependence.
doi:10.1021/bc100275r
PMCID: PMC2963316  PMID: 20879728
7.  Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages 
International journal of antimicrobial agents  2013;41(4):10.1016/j.ijantimicag.2012.11.017.
Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar–phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 μM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 μM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.
doi:10.1016/j.ijantimicag.2012.11.017
PMCID: PMC3834731  PMID: 23305655
Peptide nucleic acid (PNA); Brucella; Antibiotic resistance; Antisense; Murine macrophage
8.  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.
doi:10.1016/j.bbrc.2008.08.124
PMCID: PMC2581503  PMID: 18774771
peptide nucleic acid; antisense; anthrax protective antigen
9.  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.
doi:10.1093/nar/gkn671
PMCID: PMC2582604  PMID: 18842625
10.  Delivery of Antisense Peptide Nucleic Acids to Cells by Conjugation with Small Arginine-Rich Cell-Penetrating Peptide (R/W)9 
PLoS ONE  2014;9(8):e104999.
Peptide nucleic acids (PNAs) are very attractive antisense and antigene agents, but these molecules are not passively taken into cells. Here, using a functional cell assay and fluorescent-based methods, we investigated cell uptake and antisense activity of a tridecamer PNA that targets the HIV-1 polypurine tract sequence delivered using the arginine-rich (R/W)9 peptide (RRWWRRWRR). At micromolar concentrations, without use of any transfection agents, almost 80% inhibition of the target gene expression was obtained with the conjugate in the presence of the endosomolytic agent chloroquine. We show that chloroquine not only induced escape from endosomes but also enhanced the cellular uptake of the conjugate. Mechanistic studies revealed that (R/W)9-PNA conjugates were internalized via pinocytosis. Replacement of arginines with lysines reduced the uptake of the conjugate by six-fold, resulting in the abolition of intracellular target inhibition. Our results show that the arginines play a crucial role in the conjugate uptake and antisense activity. To determine whether specificity of the interactions of arginines with cell surface proteoglycans result in the internalization, we used flow cytometry to examine uptake of arginine- and lysine-rich conjugates in wild-type CHO-K1 and proteoglycan-deficient A745 cells. The uptake of both conjugates was decreased by four fold in CHO-745 cells; therefore proteoglycans promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Our results show that arginine-rich cell-penetrating peptides, especially (R/W)9, are a promising tool for PNA internalization.
doi:10.1371/journal.pone.0104999
PMCID: PMC4134252  PMID: 25127364
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.
doi:10.1021/bc900048y
PMCID: PMC2763590  PMID: 19678628
peptide nucleic acid; phospholipid; lipid; cell penetrating peptide; micelle; endocytosis; bioreductively cleavable
12.  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
13.  Use of Peptide Nucleic Acids to Manipulate Gene Expression in the Malaria Parasite Plasmodium falciparum 
PLoS ONE  2014;9(1):e86802.
One of the major concerns in treating malaria by conventional small drug molecules is the rapid emergence of drug resistance. Specific silencing of essential genes by antisense oliogomers has been proposed as an alternative approach that may result in antimalarial activity which is not associated with drug resistance. In addition, such an approach could be an important biological tool for studying many genes' function by reverse genetics. Here we present a novel methodology of using peptide nucleic acids (PNAs) as a useful tool for gene silencing in Plasmodium falciparum. PNAs, designed as specific antisense molecules, were conjugated to a cell penetrating peptide (CPP); namely, octa-D-lysine via the C-terminus, to allow facile delivery through cell membranes. PNAs added to P. falciparum cultures were found exclusively in infected erythrocytes and were eventually localized in nuclei of the parasites at all stages of intra erythrocytic development. We show that these PNAs specifically down regulated both a stably expressed transgene as well as an endogenous essential gene, which significantly reduced parasites' viability. This study paves the way for a simple approach to silence a variety of P. falciparum genes as means of deciphering their function and potentially to develop highly specific and potent antimalarial agents.
doi:10.1371/journal.pone.0086802
PMCID: PMC3899306  PMID: 24466246
14.  Cellular Uptakes, Biostabilities and Anti-miR-210 Activities of Chiral Arginine-PNAs in Leukaemic K562 Cells 
Chembiochem  2012;13(9):1327-1337.
A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R8-conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 m urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding.
doi:10.1002/cbic.201100745
PMCID: PMC3401907  PMID: 22639449
cell permeation; cellular differentiation; chiral PNA; microRNA; peptide nucleic acids; RNA
15.  Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions 
BMC Cancer  2010;10:342.
Background
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.
Methods
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).
Results
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.
Conclusion
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.
doi:10.1186/1471-2407-10-342
PMCID: PMC2910690  PMID: 20591158
16.  Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA. 
Nucleic Acids Research  1997;25(11):2167-2173.
We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investigated. We found that a bis-PNA (parallel antisense 10mer linked to antiparallel antisense 10mer) was superior to both the parallel antisense 10mer and antiparallel antisense 10mer in inhibiting reverse transcription of the gene, thus indicating triplex formation at the target sequence. A complete arrest of reverse transcription was obtained at approximately 6-fold molar excess of the bis-PNA with respect to the gag RNA. At this molar ratio we found no effect on in vitro translation of gag RNA. A 15mer duplex-forming PNA was also found to inhibit reverse transcription at very low molar ratios of PNA/ gag RNA. Specificity of the inhibition of reverse transcription by PNA was confirmed by RNA sequencing, which revealed that all tested RTs were stopped by the PNA/RNA complex at the predicted site. We propose that the effect of PNA is exclusively due to steric hindrance, as we found no signs of RNA degradation that would indicate PNA-mediated RNase H activation of the tested RTs. In conclusion, PNA appears to have a potential to become a specific and efficient inhibitor of reverse transcription in vivo , provided sufficient intracellular levels are achievable.
PMCID: PMC146729  PMID: 9153317
17.  Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs 
Nucleic Acids Research  2001;29(19):3965-3974.
The antisense activity of oligomers with 2′-O-methyl (2′-O-Me) phosphorothioate, 2′-O-methoxyethyl (2′-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2′-O-Me and 2′-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of l-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.
PMCID: PMC60237  PMID: 11574678
18.  Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos 
BMC Biotechnology  2012;12:50.
Background
Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications.
Results
We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes.
Conclusions
PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.
doi:10.1186/1472-6750-12-50
PMCID: PMC3469332  PMID: 22901024
PNA; Knock down; Medaka; Six3
19.  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.
doi:10.4161/adna.25628
PMCID: PMC3771998  PMID: 23954968
CCR5; PEG; PNA; antisense; nanoparticle; γPNA
20.  Targeting Listeria Monocytogenes rpoA and rpoD Genes Using Peptide Nucleic Acids 
Nucleic Acid Therapeutics  2013;23(5):363-367.
Treating intracellular pathogens remains a considerable medical challenge because of the inefficient intracellular delivery of antimicrobials and the frequent emergence of bacterial resistance to therapeutic agents deemed the drugs of last resort. We investigated the capability of antisense peptide nucleic acids (PNAs) conjugated to the (KFF)3K cell penetrating peptide to target RNA polymerase α subunit (rpoA) and RNA polymerase sigma 70 (rpoD) in the intracellular pathogen Listeria monocytogenes. The PNAs tested displayed a concentration dependent inhibition of L. monocytogenes growth in pure culture at the micromolar level and significantly reduced intracellular L. monocytogenes in infected cell culture and Caenorhabditis elegans whole animal model. In vitro, the combined PNAs treatment was synergistic resulting in a clearance of L. monocytogenes at 0.5× the individual PNA concentration. This study demonstrates the potential of anti-rpoA PNA as an antibacterial agent and will provide the basis for improving and developing these PNAs to better target intracellular pathogens like Listeria. This study also establishes C. elegans as a potential model for the screening of PNAs.
doi:10.1089/nat.2013.0426
PMCID: PMC3760085  PMID: 23859300
21.  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
22.  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.
doi:10.1093/nar/gkl249
PMCID: PMC1456331  PMID: 16670431
23.  Inhibition of Epithelial Mesenchymal Transition (EMT) With Immunochemogene Treatment in Metastatic Colorectal Cancer 
Background:
Epithelial to mesenchymal transition (EMT) causes resistance to epidermal growth factor receptor (EGFR) inhibitors. We used immunochemogene treatment composed of a stealth nanoparticle formulation, consisting of clamp PNA against mRNA of FOXC2, anti-CD44 chimeric MAb, and vinorelbine, in an attempt to eradicate metastatic colorectal cancer (mCRC) cells and inhibit metastasis by blocking EMT.
Methods:
Tumor cells from patients with stage IV chemoresistant CRC characterized by upregulation of FOXC2, CD44, and bcl-2 were obtained surgically. We synthesized antisense clamp peptide nucleic acid (PNA) oligomers (DNA analogs), in which the 6 mer homopyrimidine triplex [(PNA)2/RNA)] hybridized to the 5-end (Leader), and the 10 mer purine/pyrimidine duplex (PNA/RNA) hybridized to the 3-end (Trailer) of the AUG start codon region on the mRNA of FOXC2. The uncharged and hydrophilic antisense clamp PNA anti-FOXC2 was incorporated in the polar phase, and the vinorelbine molecules were entrapped in the acyl-chains of the lipid phase. This was surrounded by the stealth/biocompatibility polymer layer and biological recognition layer with linked chimeric MAbs against CD44 of the nanoparticle formulation. This was used to treat xenograft animal models developed from CRC cells obtained from the stage IV patients. Tumor cells were analyzed with microarray, single-nucleotide polymorphism (SNP) assay, polymerase chain reaction (PCR), western blot (WB), Southern blot (SB), immunoblotting (LC-MS/MS), immunofluorescence staining, immunohistochemistry (IHC), fluorescent activated cell sorter (FACS), confocal microscopy, transmission electron microscopy (TEM), bromodeoxyuridine (BrdU), MTT, and flow cytometry.
Results:
Post-treatment, we observed downregulation of CD44 and Fra-2, and induction of antibody-dependent cellular cytotoxicity (ADCC). The clamp PNA inhibited translation of FOXC2, resulting in activation of Jak2/Stat5a genes, which led to suppression of EMT of cancer cells. This blocked CRC metastatic invasion by reversing the mesenchymal phenotype; reconstituted homotypic adhesion; and promoted differentiation in CRC cells. Undifferentiated epithelial cells undergoing EMT exhibited overexpression of FOXC2, and this expression was lost when these cells returned to their initial differentiated epithelial state, blocking invasion and metastasis. Inhibition of EMT downregulated EGFR and inactivated NF-kB, inhibiting its downstream signaling pathway. Epithelial cell junction proteins claudin 4, claudin 7, and E-cadherin were overexpressed, upregulating beta-catenin; while mesenchymal markers vimentin and fibronectin were downregulated. Downregulation of Twist, Snail, and transcription 3 and 5 blocked the migratory potential of tumor cells, inhibiting metastasis. Calcium-independent cell-cell adhesion molecules EpCAM and TROP2 were upregulated. Vinorelbine blocked tumor cells at G2/M cell cycle, and phosphorylated bcl-2. This circumvented resistance to anoikis, inducing apoptosis in tumor cells due to lack of adhesion, inhibiting invasion and metastasis. In addition to the induction of caspase-dependent apoptosis or programmed cell death (PCD) type I in tumor cells, bcl-2 downregulation caused release of beclin-1 and upregulation of bcl-2–interacting mediator of cell death (BIM), inducing type II PCD or autophagy. TEM exhibited bystander killing effect of tumor cells by adjacent cells, and activated phagocytic cells such as macrophages. DNA synthesis and metabolic activity of tumor cells were inhibited according to BrdU and MTT tests, respectively.
Conclusion:
This immunochemogene treatment induced epithelial differentiation by reversing the mesenchymal phenotype, promoted homotypic adhesion, inhibited the multigene signature indicative of EMT, blocking metastatic cell motility/invasiveness, and eradicated mCRC cells resistant to EGFR inhibitors by induction of PCD type-I and type-II, apoptosis and autophagy, leading to a bystander killing effect.
PMCID: PMC3056306
24.  Dual Peptide Nucleic Acid- and Peptide-functionalized Shell Crosslinked Nanoparticles Designed to Target mRNA toward the Diagnosis and Treatment of Acute Lung Injury 
Bioconjugate Chemistry  2012;23(3):574-585.
In this work, multi-functional bio-synthetic hybrid nanostructures were prepared and studied for their potential utility in the recognition and inhibition of mRNA sequences for inducible nitric oxide synthase (iNOS), which are overexpressed at sites of inflammation, such as in cases of acute lung injury. Shell crosslinked knedel-like polymer nanoparticles (SCKs) that present peptide nucleic acids, for binding to complementary mRNAs, and cell penetrating peptides (CPPs), to gain cell entry, along with fluorescent labels and sites for radiolabeling, were prepared by a series of robust, efficient and versatile synthetic steps that proceeded from monomers to polymers to functional nanoparticles. Amphiphilic block graft copolymers having combinations of methoxy- and thioacetyl-terminated poly(ethylene glycol) (PEG) and DOTA-lysine units grafted from the backbone of poly(acrylic acid) (PAA) and extending with a backbone segment of poly(octadecyl acrylate-co-decyl acrylate) (P(ODA-co-DA)) were prepared by a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and chemical modification reactions, which were then used as the building blocks for the formation of well-defined SCKs decorated with reactive thiols accessible to the surface. Fluorescent labeling with Alexa Fluor 633 hydrazide was then accomplished by amidation with residual acrylic acid residues within the SCK shells. Finally, the PNAs and CPP units were covalently conjugated to the SCKs via Michael addition of thiols on the SCKs to maleimide units on the termini of PNAs and CPPs. Confirmation of the ability of the PNAs to bind selectively to the target iNOS mRNAs when tethered to the SCK nanoparticles was determined by in vitro competition experiments. When attached to the SCKs having a hydrodynamic diameter of 60 ± 16 nm, the Kd values of the PNAs were ca. an order of magnitude greater than the free PNAs, while the mismatched PNA showed no significant binding.
doi:10.1021/bc200629f
PMCID: PMC3321742  PMID: 22372643
25.  Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA 
Nucleic Acids Research  2007;35(12):3907-3917.
DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.
doi:10.1093/nar/gkm374
PMCID: PMC1919497  PMID: 17537815

Results 1-25 (734607)