Peptide Nucleic Acid Targeting Research Articles

Peptide nucleic acids (PNAs) control function of SARS-CoV-2 frameshifting stimulatory element trough PNA-RNA-PNA triplex formation

The highly structured nature of the SARS-CoV-2 genome provides many promising antiviral drug targets. One particularly promising target is a cis-acting RNA pseudoknot found within a critical region called the frameshifting stimulatory element (FSE). In this study, peptide nucleic acids (PNAs) binding to stem 2 of FSE RNA inhibited protein translation and frameshifting, as measured by a cell-free dual luciferase assay, more effectively than PNAs binding to stem 1, stem 3, or the slippery site. Surprisingly, simple antisense PNAs were stronger disruptors of frameshifting than PNA tail-clamps, despite higher thermal stability of the PNA-RNA-PNA triplexes formed by the latter. Another unexpected result was a strong and sequence non-specific enhancement of frameshifting inhibition when using a cationic triplex-forming PNA in conjunction with an antisense PNA targeting key regions of the frameshifting element. Our results illustrate both the potential and the challenges of using antisense PNAs to target highly structured RNAs, such as SARS-CoV-2 pseudoknots. While triplex forming PNAs, including PNA tail-clamps, are emerging as promising ligands for RNA recognition, the binding affinity enhancements when using cationic modifications in triplex-forming PNAs must be carefully balanced to avoid loss of sequence specificity in complex biological systems.

Carrier capability of halloysite nanotubes for the intracellular delivery of antisense PNA targeting mRNA of neuroglobin gene

Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently.Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis.

Treatment of Human Glioblastoma U251 Cells with Sulforaphane and a Peptide Nucleic Acid (PNA) Targeting miR-15b-5p: Synergistic Effects on Induction of Apoptosis.

Glioblastoma multiforme (GBM) is a lethal malignant tumor accounting for 42% of the tumors of the central nervous system, the median survival being 15 months. At present, no curative treatment is available for GBM and new drugs and therapeutic protocols are urgently needed. In this context, combined therapy appears to be a very interesting approach. The isothiocyanate sulforaphane (SFN) has been previously shown to induce apoptosis and inhibit the growth and invasion of GBM cells. On the other hand, the microRNA miR-15b is involved in invasiveness and proliferation in GBM and its inhibition is associated with the induction of apoptosis. On the basis of these observations, the objective of the present study was to determine whether a combined treatment using SFN and a peptide nucleic acid interfering with miR-15b-5p (PNA-a15b) might be proposed for increasing the pro-apoptotic effects of the single agents. To verify this hypothesis, we have treated GMB U251 cells with SFN alone, PNA-a15b alone or their combination. The cell viability, apoptosis and combination index were, respectively, analyzed by calcein staining, annexin-V and caspase-3/7 assays, and RT-qPCR for genes involved in apoptosis. The efficacy of the PNA-a15b determined the miR-15b-5p content analyzed by RT-qPCR. The results obtained indicate that SFN and PNA-a15b synergistically act in inducing the apoptosis of U251 cells. Therefore, the PNA-a15b might be proposed in a “combo-therapy” associated with SFN. Overall, this study suggests the feasibility of using combined treatments based on PNAs targeting miRNA involved in GBM and nutraceuticals able to stimulate apoptosis.

Extracellular vesicles mediated exocytosis of antisense peptide nucleic acids

Peptide nucleic acids (PNAs), a synthetic DNA mimic, have been extensively utilized for antisense- and antigene-based biomedical applications. Significant efforts have been made to increase the cellular uptake of PNAs, but here we examined relatively unexplored aspects of intracellular trafficking and endocytic recycling of PNAs. For proof-of-concept, we used anti-microRNA (miR) PNA targeting miR-155. The sub-cellular localization of PNA was studied via confocal and flow-cytometry-based assays in HeLa cells. A comprehensive characterization of PNA-containing extracellular vesicles revealed spherical morphology, negative surface charge density, and the presence of tetraspanin markers. Most importantly, we investigated rab11a and rab27b GTPases’ role in regulating the exocytosis of PNAs. Organelle staining, followed by confocal imaging, showed higher localization of PNA in lysosomes. Gene-expression analysis established the enhanced functional activity of PNA after inhibition of endocytic recycling. Multiple studies report the exocytosis of single-stranded oligonucleotides, short interfering RNAs (siRNAs), and nanocarriers. To our knowledge, this is the first mechanistic study to establish that PNA undergoes endocytic recycling and exocytosis out of tumor cells. The results presented here can serve as a platform to develop and optimize strategies for improving the therapeutic efficacy of PNAs by avoiding the recycling pathways.

Synergistic effects of the combined treatment of U251 and T98G glioma cells with an anti‑tubulin tetrahydrothieno[2,3‑c]pyridine derivative and a peptide nucleic acid targeting miR‑221‑3p.

In the development of novel and more effective anti-cancer approaches, combined treatments appear to be of great interest, based on the possibility of obtaining relevant biological or therapeutic effects using lower concentrations of single drugs. Combination therapy may prove to be of utmost significance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer cases of the central nervous system, with a median survival rate of 15 months. As regards novel therapeutic approaches, the authors have recently demonstrated that peptide nucleic acids (PNAs) that target microRNA (miRNA/miR)-221 are very active in inducing the apoptosis of glioma cells. Furthermore, in a recent study, the authors described two novel series of tubulin polymerization inhibitors based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene scaffold, which exerted a potent anti-proliferative effect on a variety of tumor cell lines. The present study aimed to verify the activity on glioblastoma cancer cell lines of one of the most active compounds tested, corresponding to 2-(3′, 4′, 5′-trimethoxyanilino)-3-cyano/alkoxycarbonyl-6-substituted-4 5,6,7-tetrahydrothiene[2,3-c] pyridine (compound 3b), used in combination with an anti-miR-221-3p PNA, already demonstrated to be able to induce high levels of apoptosis. To the best of our knowledge, the results obtained herein demonstrate for the first time a 'combination therapy' performed by the combined use of a PNA targeting miR-221 and the tetrahydrothiene[2,3-c]pyridine derivative 3b, supporting the concept that the combined treatment of GBM cells with a PNA against a specific upregulated oncomiRNA (in the present study a PNA targeting miR-221-3p was used) and anti-tubulin agents (in the present study derivative 3b was used) is an encouraging strategy which may be used to enhance the efficacy of anticancer therapies and at the same time, to reduce side-effects.

A Peptide-Nucleic Acid Targeting miR-335-5p Enhances Expression of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene with the Possible Involvement of the CFTR Scaffolding Protein NHERF1.

(1) Background: Up-regulation of the Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) might be of great relevance for the development of therapeutic protocols for cystic fibrosis (CF). MicroRNAs are deeply involved in the regulation of CFTR and scaffolding proteins (such as NHERF1, NHERF2 and Ezrin). (2) Methods: Content of miRNAs and mRNAs was analyzed by RT-qPCR, while the CFTR and NHERF1 production was analyzed by Western blotting. (3) Results: The results here described show that the CFTR scaffolding protein NHERF1 can be up-regulated in bronchial epithelial Calu-3 cells by a peptide-nucleic acid (PNA) targeting miR-335-5p, predicted to bind to the 3′-UTR sequence of the NHERF1 mRNA. Treatment of Calu-3 cells with this PNA (R8-PNA-a335) causes also up-regulation of CFTR. (4) Conclusions: We propose miR-335-5p targeting as a strategy to increase CFTR. While the efficiency of PNA-based targeting of miR-335-5p should be verified as a therapeutic strategy in CF caused by stop-codon mutation of the CFTR gene, this approach might give appreciable results in CF cells carrying other mutations impairing the processing or stability of CFTR protein, supporting its application in personalized therapy for precision medicine.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i> in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

High Levels of Apoptosis Are Induced in the Human Colon Cancer HT-29 Cell Line by Co-Administration of Sulforaphane and a Peptide Nucleic Acid Targeting miR-15b-5p.

Sulforaphane (SFN) is one of most important dietary constituents of broccoli (Brassica oleracea) and other cruciferous vegetables, which have been reported to exhibit health benefits, including prevention and therapy of cancer, such as colorectal carcinoma (CRC). The objective of this study was to determine whether the anticancer effect of SFN on colon cancer HT-29 cell line could be improved by the combined treatment with molecules inhibiting microRNAs (miRNAs) involved in CRC. As miRNA inhibiting molecules we focused on peptide-nucleic acids (PNAs). As miRNA to be targeted, miR-15b-5p was selected on the basis of several information present in the literature and confirming that miR-15b-5p is overexpressed in colon cancer patients, and that its targeting decreases cell migration and metastasis in colorectal cancer. In this article, we described for the first time the efficacy of targeting miR-15b-5p by using a PNA against miR-15b-5p (R8-PNA-a15b), functionalized with an octoarginine peptide (R8) for maximizing cellular uptake. The miR-15b-5p downregulation in the colon cancer HT-29 cell line was associated with inhibition of in vitro cell growth and activation of the proapoptotic pathway, demonstrated by a sharp increase of late apoptotic cells in HT-29-treated cell populations. A second conclusion of this study is that the R8-PNA-a15b might be proposed in "combo-therapy" associated with SFN. To our knowledge, no report is available in the literature on a combination between SFN and miRNA-targeting molecules. Our data demonstrate that this combined treatment leads to a very high proportion of apoptotic HT-29 cells (over 85%), a value higher than the sum of the values of apoptotic cells obtained after singularly administered regents (either SFN or R8-PNA-a15b).

Antibacterial Peptide Nucleic Acids-Facts and Perspectives.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.

Demonstrating specificity of bioactive peptide nucleic acids (PNAs) targeting microRNAs for practical laboratory classes of applied biochemistry and pharmacology.

Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis.

Developments in Cell-Penetrating Peptides as Antiviral Agents and as Vehicles for Delivery of Peptide Nucleic Acid Targeting Hepadnaviral Replication Pathway.

Alternative therapeutic approaches against chronic hepatitis B virus (HBV) infection need to be urgently developed because current therapies are only virostatic. In this context, cell penetration peptides (CPPs) and their Peptide Nucleic Acids (PNAs) cargoes appear as a promising novel class of biologically active compounds. In this review we summarize different in vitro and in vivo studies, exploring the potential of CPPs as vehicles for intracellular delivery of PNAs targeting hepadnaviral replication. Thus, studies conducted in the duck HBV (DHBV) infection model showed that conjugation of (D-Arg)8 CPP to PNA targeting viral epsilon (ε) were able to efficiently inhibit viral replication in vivo following intravenous administration to ducklings. Unexpectedly, some CPPs, (D-Arg)8 and Decanoyl-(D-Arg)8, alone displayed potent antiviral effect, altering late stages of DHBV and HBV morphogenesis. Such antiviral effects of CPPs may affect the sequence-specificity of CPP-PNA conjugates. By contrast, PNA conjugated to (D-Lys)4 inhibited hepadnaviral replication without compromising sequence specificity. Interestingly, Lactose-modified CPP mediated the delivery of anti-HBV PNA to human hepatoma cells HepaRG, thus improving its antiviral activity. In light of these promising data, we believe that future studies will open new perspectives for translation of CPPs and CPP-PNA based technology to therapy of chronic hepatitis B.

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

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

Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)n Repeats by PNA or LNA Targeting.

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.

Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

Ultrasound microbubble-carried PNA targeting to c-myc mRNA inhibits the proliferation of rabbit iliac arterious smooth muscle cells and intimal hyperplasia

Objective: To elucidate the transfected effect of albumin ultrasound microbubbles carrying peptide nucleic acids (PNAs) against c-myc gene to the vascular walls and their effect on the intimal proliferation induced by vascular denudation.Methods: A rabbit iliac artery intimal proliferation model was constructed and PNA against c-myc mRNA was designed and synthesized and was added to albumin solution before ultrasound microbubbles were prepared and encapsulated in matrix of albumin. The ultrasound microbubbles carrying PNA were transfected to intima under ultrasound exposure. The transfected effect was identified by a histochemical method and the expression of c-myc was detected by in situ hybridization. The proliferation of intimal smooth muscle cells was estimated by the expression of proliferative cell nuclear antigen (PCNA) of them. The intimal area and thickness were judged morphologically for intimal hyperplasia.Results: The ultrasound microbubbles with PNA were successfully prepared and c-myc PNA was transfected to vascular intimal cells. The expression of c-myc and PCNA by intimal vascular smooth muscle cells (vSMCs) was inhibited significantly and the intimal thickness and area were reduced remarkably.Conclusion: Transfection of c-myc PNA could inhibit proliferartion of vSMCs and intima in the rabbit iliac artery intimal proliferation model and the targeted transfection of albumin ultrasound microbubbles carrying PNA offers a feasible way to facilitate its access to specific cells in vivo and produce bioavailability.

A Peptide Nucleic Acid to Reduce Type I Collagen Production by Fibroblast Cells

In this study, we prepared a peptide nucleic acid (PNA) against the gene coding for the human alpha 1 chain of type I collagen. This PNA was incorporated into normal human fibroblast cells by electroporation, leading to a decrease in the mRNA level of the gene. Furthermore, mRNA for the alpha 2 chain of type I collagen was also reduced. The production of collagen protein exhibited a similar profile to the changes in mRNA. These results indicate that PNA targeting COL1A1 is effective as an antigene reagent, and opens the possibility of future clinical applications in fibroproliferative disorders.

A Peptide Nucleic Acid Targeting Nuclear Rad51 Sensitizes Myeloma Cells to Melphalan Chemotoxicity Both in Vitro and in Vivo

Multiple myeloma (MM) cells are characterized by extensive genomic heterogeneity, which contributes to patient differences in prognosis and response to treatment. We previously reported that MM cells have elevated homologous recombination (HR) rates and expression of RAD51 and its paralogs, promoting genomic instability and disease progression that are reversed by RAD51 siRNA. We now examine the roles of HR and RAD51 in resistance to melphalan, one of the most widely used drugs for MM chemotherapy. The drug induces a variety of DNA lesions, with DNA interstrand crosslinks (ICL) accounting for most of the drug’s cytotoxicity. RAD51 is a central protein in the HR pathway and its overexpression may contribute to chemoresistance by enabling repair of DNA lesions induced by DNA damaging agents such as melphalan. MM cell sensitivity to melphalan correlates directly with melphalan-induced RAD51 foci, and high RAD51 expression predicts poor event-free and overall survival of MM patients. Activity of the Rad51 promoter increases >850-fold in cancer cells compared to normal cells, and tumor cells are selectively killed by a construct in which PRad51 drives expression of diphtheria toxin. In this study, we tested whether inhibiting RAD51 expression with a peptide nucleic acid (PNA) would inhibit MM cell growth and/or sensitize MM cells to melphalan. PNAs are DNA or RNA mimics in which a polymer of (2-amino­ethyl) glycine replaces the nucleic acid’s sugar-phosphate backbone. PNAs are highly specific, binding DNA with higher affinity than RNA or DNA, and they are quite stable to degradation both in vitro and in vivo.We designed a PNA to target the promoter region of the RAD51 gene (PNArad51), encompassing the transcription start site. To enhance cellular uptake and nuclear delivery without transfection, we conjugated the PNA to a nuclear localization signal rich in basic residues (PKKKRKVR). As a control we employed a scrambled PNA (PNAmt) with the same nucleotide composition but not targeting any genomic sequences. We used qRT-PCR to assess the effect of PNA on RAD51 mRNA expression and that of melphalan on mRNA levels of RAD51 and its paralogs (RAD51B, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) and BRCA1. Propidium iodide staining and flow cytometry were used to examine the cell-cycle effects of melphalan. γH2AX and RAD51 foci were quantitated using confocal immuno­fluorescence microscopy, and MM cell viability was assessed with the WST-1 assay. To examine the in vivo consequences of PNA ± melphalan for tumor growth, we injected H929 MM cells expressing luciferase into rabbit bone fragments implanted in SCID-rab mice, as previously described by us,. Total RNA extracted from cells recovered from the rabbit bones was analyzed by qRT-PCR to determine the in vivo effect of PNA on expression of RAD51.Melphalan treatment (10 µM) significantly induced expression of RAD51 and its paralogs, particularly RAD51 and XRCC3 (p≤0.01). Melphalan caused cell-cycle arrest, predominantly in the S-phase (55%, significantly elevated over vehicle alone, 17%; p<0.0001), the period in which HR is most active, and during which ICLs are converted into double strand breaks (DSBs) on encountering DNA replication forks. PNArad51 (10 µM) significantly reduced expression of RAD51 (~60%, p<0.001) relative to PNAmt. Pretreatment with PNArad51 inhibited melphalan-induced RAD51 focus formation, far more than PNAmt pretreatment (21% compared to 66%, p<0.0001) whereas the number of γH2AX foci increased (66%) relative to PNAmt (39%; p<0.0001). Consequently, pretreatment with PNArad51 produced synergistic synthetic lethality with melphalan, reducing the IC50 of melphalan by 4.5-fold. PNArad51 alone caused significant cytotoxicity compared to PNAmt (p<0.05). In the SCID-rab mouse model, a two-week treatment with PNArad51 alone or in combination with melphalan resulted in significant inhibition of tumor volume (p≈0.01 and p<0.05, respectively) compared to PNAmt, although the combination of PNAmt plus melphalan was ineffectual. Prolonged treatment (4 weeks) with PNArad51 ± melphalan (but not PNAmt + melphalan) reduced tumor growth compared to PNAmt treatment, although this was not statistically significant (p>0.05).These results highlight the importance of RAD51 in the response of MM cells to melphalan, and indicate for the first time the potential for RAD51-targeted PNA in tumor chemosensitization. DisclosuresNo relevant conflicts of interest to declare.

Hybridization of G-Quadruplex-Forming Peptide Nucleic Acids to Guanine-Rich DNA Templates Inhibits DNA Polymerase η Extension

The guanine quadruplex (G-quadruplex) is a highly stable secondary structure that forms in G-rich repeats of DNA, which can interfere with DNA processes, including DNA replication and transcription. We showed previously that short guanine-rich peptide nucleic acids (PNAs) can form highly stable hybrid quadruplexes with DNA. We hypothesized that such structures would provide a stronger block to polymerase extension on G-rich templates than a native DNA homoquadruplex because of the greater thermodynamic stability of the PNA-DNA hybrid structures. To test this, we analyzed the DNA primer extension activity of polymerase η, a translesion polymerase implicated in synthesis past G-quadruplex blocks, on DNA templates containing guanine repeats. We observed a PNA concentration-dependent decrease in the level of polymerase η extension to the end of the template and an increase in the level of polymerase η inhibition at the sequence prior to the G-rich repeats. In contrast, the addition of a complementary C-rich PNA that hybridizes to the G-rich repeats by Watson-Crick base pairing led to a decrease in the level of polymerase inhibition and an increase in the level of full-length extension products. The G-quadruplex-forming PNA exhibited inhibition (IC50=16.2±3.3 nM) of polymerase η DNA synthesis on the G-rich templates stronger than that of the established G-quadruplex-stabilizing ligand BRACO-19 (IC50=42.5±4.8 nM). Our results indicate that homologous PNA targeting of G-rich sequences creates stable PNA-DNA heteroquadruplexes that inhibit polymerase η extension more effectively than a DNA homoquadruplex. The implications of these results for the potential development of homologous PNAs as therapeutics for halting proliferating cancer cells are discussed.

Uptake by human glioma cell lines and biological effects of a peptide-nucleic acids targeting miR-221

MicroRNAs are a family of small noncoding RNAs regulating gene expression by sequence-selective mRNA targeting, leading to a translational repression or mRNA degradation. The oncomiR miR-221 is highly expressed in human gliomas, as confirmed in this study in samples of low and high grade gliomas, as well in the cell lines U251, U373 and T98G. In order to alter the biological functions of miR-221, a peptide nucleic acid targeting miR-221 (R8-PNA-a221) was produced, bearing a oligoarginine peptide (R8) to facilitate uptake by glioma cells. The effects of R8-PNA-a221 were analyzed in U251, U373 and T98G glioma cells and found to strongly inhibit miR-221. In addition, the effects of R8-PNA-a221 on p27(Kip1) (a target of miR-221) were analyzed in U251 and T98G cells by RT-qPCR and by Western blotting. No change of p27(Kip1) mRNA content occurs in U251 cells in the presence of PNA-a221 (lacking the R8 peptide), whereas significant increase of p27(Kip1) mRNA was observed with the R8-PNA-a221. These data were confirmed by Western blot assay. A clear increment of p27(Kip1) protein expression in the samples treated with R8-PNA-a221 was detected. In addition, R8-PNA-a221 was found able to increase TIMP3 expression (another target of miR-221) in T98G cells. These results suggest that PNAs against oncomiRNA miR-221 might be proposed for experimental treatment of human gliomas.

Species-Selective Killing of Bacteria by Antimicrobial Peptide-PNAs

Broad-spectrum antimicrobials kill indiscriminately, a property that can lead to negative clinical consequences and an increase in the incidence of resistance. Species-specific antimicrobials that could selectively kill pathogenic bacteria without targeting other species in the microbiome could limit these problems. The pathogen genome presents an excellent target for the development of such antimicrobials. In this study we report the design and evaluation of species-selective peptide nucleic acid (PNA) antibacterials. Selective growth inhibition of B. subtilis, E. coli, K. pnuemoniae and S. enterica serovar Typhimurium in axenic or mixed culture could be achieved with PNAs that exploit species differences in the translation initiation region of essential genes. An S. Typhimurium-specific PNA targeting ftsZ resulted in elongated cells that were not observed in E. coli, providing phenotypic evidence of the selectivity of PNA-based antimicrobials. Analysis of the genomes of E. coli and S. Typhimurium gave a conservative estimate of >150 PNA targets that could potentially discriminate between these two closely related species. This work provides a basis for the development of a new class of antimicrobial with a tuneable spectrum of activity.