Peptide Nucleic Acid Oligonucleotides Research Articles

Does a Conjugation Site Affect Transport of Vitamin B12 -Peptide Nucleic Acid Conjugates into Bacterial Cells?

Gram-negative bacteria develop specific systems for the uptake of scarce nutrients, including vitamin B12 . These uptake pathways may be utilized for the delivery of biologically relevant molecules into cells. Indeed, it was recently reported that vitamin B12 transported an antisense peptide nucleic acid (PNA) into Escherichia coli and Salmonella Typhimurium cells. The present studies indicate that the conjugation site of PNA to vitamin B12 has an impact on PNA transport into bacterial cells. Toward this end, a specifically designed PNA oligomer has been tethered at various positions of vitamin B12 (central Co, R5' -OH, c and e amide chains, meso position, and at the hydroxy group of cobinamide) by using known or newly developed methodologies and tested for the uptake of the synthesized conjugates by E. coli. Compounds in which the PNA oligonucleotide was anchored at the R5' -OH position were transported more efficiently than that of other compounds tethered at the peripheral positions around the corrin ring. Of importance is the fact that, contrary to mammalian organisms, E. coli also takes up cobinamide, which is an incomplete corrinoid. This selectivity opens up ways to fight bacterial infections.

Vitamin B12 as a carrier of peptide nucleic acid (PNA) into bacterial cells

Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B12 conjugates; namely the vitamin B12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B12 and PNA were tested, including a disulfide bond. We found that vitamin B12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)3K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B12 as a carrier of PNA oligonucleotides into bacterial cells.

Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences

Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (Tm) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity.

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

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

Peptide Nucleic Acid (PNA)-Clamping Competitive PCR: A New Tool for the High Sensitive Detection of JAK2V617F Mutation in Myeloproliferative Neoplasms (MPNs)

Abstract 5168 Background:Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by clonal expansion of single or multiple lineages of myeloid subset (i.e. granulocytic, erythroid, megakaryocytic and mast cell). Since 2008, the WHO included the detection of JAK2 mutations (the common V617F and the less common exon 12 mutations), into the diagnostic criteria of MPNs. The specific pathogenic implication of JAK2 mutations in MPNs is still under investigation. Preliminary data emerging from the treatment of Idiopathic Myelofibrosis patients with JAK2 inhibitors have shown only clinically significant benefits (improvement of splenomegaly and constitutional symptoms) but not a clear evidence of disease-modifying activity. Several techniques (e.g. ASO-PCR, ARMS-PCR, Direct sequencing, HRM, DHPLC, etc) have been used to detect the JAK2 V617F mutation, but all of them were low sensitive and time-consuming. We developed a PNA-clamping competitive PCR assay able to detect JAK2 V617F mutation with a very high level of sensitivity and specificity. Methods:In order to promote the selective amplification of the JAK2 V617F mutant allele, a specific PNA oligonucleotide, full matching with the wild-type (wt) sequence of the portion of JAK2 gene containing the V617F mutation, was designed to compete with the reverse primer used in the PCR assay. It was experimentally demonstrated that a PNA concentration of 6 μmol/L occurred for the complete clamping of 100 ng of wt genomic DNA used as template for the PCR reaction. The sensitivity of the assay was determined by a serial dilution (100% through 0.01%) of genomic DNA containing JAK2 V617F mutation, obtained from HEL cell line, with wt DNA obtained from healthy donors. The specificity of PCR products was assessed by sequencing in both forward and reverse directions. The thermal dissociation profile of PNA/DNA and DNA/DNA duplexes was studied by monitoring the Enthalpy as function of the temperature (range 20–100°C), with an heating/cooling rate of 1°C/min. Results:PNA-clamping competitive PCR was able to detect JAK2 V617F mutation with a sensitivity of 0.01%. Thermodynamic studies clearly showed that the melting temperature (Tm) of fully matched PNA/DNA duplex is always higher than Tm of the corresponding DNA/DNA duplex. The enthalpy values for the hybrid PNA/DNA are always greater than the corresponding DNA/DNA duplex and a single mismatch has an energetic cost higher in a PNA/DNA than in DNA/DNA duplex. This energetic cost is more evident in the melting temperature with a ΔTm of 16°C and 7°C for the PNA/DNA and DNA/DNA duplex, respectively. In the PCR assay the PNA complementary to the JAK2 wild type sequence strongly compete with the primer resulting in a complete knock out of the wild type allele. In a blind screening of samples obtained from 308 MPN patients, PNA clamping competitive PCR was able to detect JAK2 V617F mutation in 61.4% cases of Polycythemia Vera, 54.4% of Essential Thrombocythemia, 57.9% of Idiopathic Myelofibrosis and in 21.4% of Ph negative-MPNs, respectively. In 40 unselected patients, PNA clamping PCR results were confirmed by other techniques such as ASO-PCR, ARMS-PCR and direct sequencing. Conclusions:The PNA-clamping competitive PCR assay could be used as convenient, high-sensitive and reliable diagnostic test for detection of JAK2 V617F mutation both on genomic DNA and cDNA samples. As suggested from the thermodynamic studies, a similar technique could be developed for the detection of any known single point mutation, widely contributing to the improvement of molecular diagnostic tests usable in clinical practice. Disclosures:No relevant conflicts of interest to declare.

Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR : Blocking Wild-Type Amplification by Using a Peptide Nucleic Acid Oligonucleotide

A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F.

PNA-based artificial nucleases as antisense and anti-miRNA oligonucleotide agents

Because of its interesting chemical, physical and biological properties, Peptide Nucleic Acid (PNA) has attracted major attention in molecular biology, for diagnostics purposes and development of biosensors. PNAs have become candidates for gene therapeutic drugs in ANTISENSE (AO) strategy with favorable in vivo biochemical properties. Recently, antisense PNA oligonucleotides have been described in anti-miRNA approach (AMO). We propose PNA-based nucleases as AO and AMO agents. We report the design, synthesis and characterization of two kinds of artificial nucleases composed of a PEG-PNA-PEG domain conjugated to HGG·Cu (A) and DETA (B) as well known cleavage sites. Qualitative (MALDI-TOF) and quantitative (HTS) assays were planned to study nuclease activity of constructs A and B on RNA-3'-FAM target sequence. The results have highlighted the best performance of nuclease B and the relevance of the PEG spacer, in particular for conjugate A, in terms of efficiency of the cleavage, suggesting that conjugates A and B also act as potential antisense and anti-miRNA agents.

Abstract 4073: The PNA oligonucleotide is effective miRNA inhibitor that transfection reagents are not required

Abstract The miRNAs are approximately ∼23 nucleotides non-coding RNAs. They regulate major processes such as development, apoptosis, cell proliferation, hematopoiesis, and patterning of the nervous system. MiRNA inhibition using antisense oligonucleotide is an effective tool for understanding miRNA function. The peptide nucleic acid (PNA) is an artificial oligonucleotide with a peptide backbone. PNAs have a stronger affinity and greater specificity than DNA or RNA and are resistant to nuclease, which is essential characteristic for a miRNA inhibitor that will be exposed to abundant serum and cellular nucleases. In here, we evaluated PNAs™ miRNA inhibitors in cultured cells including stem cell using luciferase assay and qPCR. FACS analysis was showed that PNAs™ miRNA inhibitors can penetrate to intra cells without transfection reagent. Also, miRNA inhibition effects are sustained until 15 days after treatment. PNAs™ miRNA inhibitor has a lower cytotoxicity than other nucleic acid-based miRNA inhibitors. PNAs™ miRNA inhibitors can be stored at room temperature for 20 weeks without losing the activity. These results demonstrate that PNAs are a powerful and effective tool to studies of miRNA mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4073.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

Taking control of gene expression with light-activated oligonucleotides

The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.

Antisense PNA Accumulates in Escherichia coli and Mediates a Long Post-antibiotic Effect

Antisense agents that target growth-essential genes display surprisingly potent bactericidal properties. In particular, peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomers linked to cationic carrier peptides are effective in time kill assays and as inhibitors of bacterial peritonitis in mice. It is unclear how these relatively large antimicrobials overcome stringent bacterial barriers and mediate killing. Here we determined the transit kinetics of peptide-PNAs and observed an accumulation of cell-associated PNA in Escherichia coli and slow efflux. An inhibitor of drug efflux pumps did not alter peptide-PNA potency, indicating a lack of active efflux from cells. Consistent with cell retention, the post-antibiotic effect (PAE) of the anti-acyl carrier protein (acpP) peptide-PNA was greater than 11 hours. Bacterial cell accumulation and a long PAE are properties of significant interest for antimicrobial development.

RNA hairpin invasion and ribosome elongation arrest by mixed base PNA oligomer.

Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha-ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha-ras mRNA rests upon the stability of PNA-mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA-RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C.G*C(+) and T.A*T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA-RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA-RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA-RNA complexes using matrix-assisted laser desorption/ionization time-of -flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes.

Quantitative Fluorescence In Situ Hybridization (Q‐FISH)

This unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide "mimetic" which has a higher affinity than regular oligonucleotide (RNA or DNA) probes for complementary single-strand (ss) DNA sequences. PNA oligonucleotides have excellent penetration properties due to their small size (typically 15 to 18-mers) and can be directly labeled with fluorochromes. These properties have been exploited to develop quantitative fluorescence in situ hybridization (Q-FISH) onto denatured single-stranded chromosomal DNA target sequences. The latter can be present in preparations of fixed metaphase cells on slides (Q-FISH) or in heat-treated (interphase) cells in suspension (flow-FISH).

Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water

A species-specific Peptide Nucleic Acid (PNA) oligonucleotide probe directed against the V1region of the 16S rRNA molecule was synthesized for the detection of Escherichia coli in water. The specificity of the probe was tested in dot blot hybridizations against a number of environmental isolates including those from the genera Escherichia , Klebsiella , Enterobacter and Citrobacter . In situ hybridization experiments were performed with biotinylated PNA oligonucleotide probes with subsequent detection of the biotin label using a combination of Streptavidin-Horseradish Peroxidase and a tyramide signal amplification system. The results obtained enabled the specific detection of E. coli in under 3 h. Hybridizations were also performed on cells which were treated with chlorine (1·5 mg l−1) for up to 30 min. Escherichia coli cells were still detected after storage for 14 days at room temperature. No cells were detected by conventional plate count or the «Colilert» assay, a method currently used for the routine detection of E. coli and coliforms in the water industry. Cell viability was assessed by the ability of cells to reduce 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to highly fluorescent formazan crystals through bacterial respiration. Only cells that had not been chlorinated were detected. These results confirm that ribosomal RNA exists within the cell long after cell death has occurred and that rRNA cannot be used to assess the viability of individual cells. However rRNA probes in combination with viability markers should enable the specific detection of viable cells in situ . Hybridization experiments were also performed successfully on seeded tap water samples. The number of fluorescent cells detected correlated well with those obtained by plate count analysis. This represents the first reported use of PNA oligonucleotides forin situ detection of micro-organisms and offers a fast efficient alternative to conventional DNA approaches.

Template switching between PNA and RNA oligonucleotides.

The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.