Search

Background: Staphylococcus aureus and S. pseudintermedius are the major etiological agents of staphylococcal infections in humans, livestock, and companion animals. The misuse of antimicrobial drugs has led to the emergence of antimicrobial-resistant Staphylococcus spp., including methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP). One novel therapeutic approach against MRSA and MRSP is a peptide nucleic acid (PNA) that can bind to the target nucleotide strands and block expression. Previously, two PNAs conjugated with cell-penetrating peptides (P-PNAs), antisense PNA (ASP)-cmk and ASP-deoD, targeting two essential genes in S. aureus, were constructed, and their antibacterial activities were analyzed. Objectives: This study analyzed the combined antibacterial effects of P-PNAs on S. aureus and S. pseudintermedius clinical isolates. Methods: S. aureus ATCC 29740 cells were treated simultaneously with serially diluted ASPcmk and ASP-deoD, and the minimal inhibitory concentrations (MICs) were measured. The combined P-PNA mixture was then treated with S. aureus and S. pseudintermedius veterinary isolates at the determined MIC, and the antibacterial effect was examined. Results: The combined treatment of two P-PNAs showed higher antibacterial activity than the individual treatments. The MICs of two individual P-PNAs were 20 and 25 μM, whereas that of the combined treatment was 10 μM. The application of a combined treatment to clinical Staphylococcus spp. revealed S. aureus isolates to be resistant to P-PNAs and S. pseudintermedius isolates to be susceptible. Conclusions: These observations highlight the complexity of designing ASPs with high efficacy for potential applications in treating staphylococcal infections in humans and animals. [ABSTRACT FROM AUTHOR]

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid (PNA) derivatives, including eleven PNA monomers and two PNA decamers, is described. The acidity constants (pK a) of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution, and these values were found to be in agreement with those obtained by MoKa software. These results indicate that single nucleobases do not change their pK a values when included in PNA monomers and oligomers. In addition, immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers, which showed the PNA derivatives had poor affinity towards membrane phospholipids, and confirmed their scarce cell penetrating ability. Overall, our study not only is of potential relevance to evaluate the pharmacokinetic properties of PNA, but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties. [Display omitted] • The first study on acid-base and lipophilic properties of peptide nucleic acids (PNA). • pK a of acid-base centers of PNA evaluated by potentiometric method and MoKa prediction. • NMR experiments provide additional information on the protonation of PNA monomers. • Lipophilicity of PNA monomers and oligomers is investigated by IAM chromatography. • This study can lay the basis of evaluating the pharmacokinetic properties of PNA. [ABSTRACT FROM AUTHOR]

Abstract FISH-based RNA detection in paraffin-embedded tissue can be challenging, with complicated procedures producing uncertain results and poor image quality. Here, we developed a robust RNA detection method based on graphene oxide (GO) quenching and recovery of fluorescence in situ hybridization (G-FISH) in formalin-fixed paraffin-embedded (FFPE) tissues. Using a fluorophore-labeled peptide nucleic acid (PNA) attached to GO, the endogenous long noncoding RNA BC1, the constitutive protein β-actin mRNA, and miR-124a and miR-21 could be detected in the cytoplasm of a normal mouse brain, primary cultured hippocampal neurons, an Alzheimer's disease model mouse brain, and glioblastoma multiforme tumor tissues, respectively. Coding and non-coding RNAs, either long or short, could be detected in deparaffinized FFPE or frozen tissues, as well as in clear lipid-exchanged anatomically rigid imaging/immunostaining-compatible tissue hydrogel (CLARITY)-transparent brain tissues. The fluorescence recovered by G-FISH correlated highly with the amount of miR-21, as measured by quantitative real time RT-PCR. We propose G-FISH as a simple, fast, inexpensive, and sensitive method for RNA detection, with a very low background, which could be applied to a variety of research or diagnostic purposes. Graphical Abstract Fluorescence in situ hybridization (FISH) method for RNA detection in paraffin-embedded tissue contains complicated protocols with poor image quality. In this study, we developed a robust nanoplatform using graphene oxide (GO) quenching and recovery of fluorescence in situ hybridization (G-FISH) for sensitive RNA detection in formalin-fixed paraffin-embedded (FFPE) tissues. A carboxyfluorescein (FAM) dye-decorated peptide nucleic acid (PNA) probe complementary to the target RNA was attached to graphene oxide. Rapid hybridization between the FAM-PNA probes and target RNAs leads to recovery of fluorescence signals in FFPE tissue section. Brain specific BC1 long noncoding RNA and miR-124a expression was markedly detected in paraffin-embedded brain tissue. The difference in BC1 expression was seen in brain section of Alzheimer's disease model. G-FISH also showed highly expressed miR-21 level in human glioblastoma brain tissue sample. Unlabelled Image [ABSTRACT FROM AUTHOR]

Purpose: Molecular testing in non-small cell lung cancer (NSCLC) aids in identifying oncogenic alterations. The aim of this study was to compare the rates of detection of oncogenic alterations and responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) according to EGFR mutation status as determined by peptide nucleic acid (PNA) clamping or direct sequencing (DS). Materials and Methods: We performed a systematic literature search using MEDLINE, EMBASE, and the Cochrane Central Register. Data from included studies were pooled to yield summary sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and receiver operating characteristic curves. A meta-regression analysis was conducted to identify potential sources of heterogeneity between selected studies. Results: We identified 10 studies comprising 924 patients. Oncogenic alterations were detected in 340 of 924 cases (36.8%) with PNA clamping and in 250 of 924 (27.1%) with DS. The pooled sensitivities of PNA clamping and DS were 0.93 [95% confidence interval (CI): 0.90-0.95] and 0.69 (95% CI: 0.64-0.73), respectively. According to meta-regression analysis, none of the covariates were found to be significant sources of heterogeneity. With respect to treatment responses to EGFR-TKIs, there was no significant difference therein between EGFR mutations detected by PNA clamping and DS (53.4% vs. 50.8%; risk ratio, 0.99; 95% CI 0.83-1.19; p=0.874). Conclusion: We demonstrated that PNA clamping has a higher sensitivity than DS for detecting oncogenic alterations in NSCLC. Our findings suggest that PNA clamping is a more useful method for clinical practice. [ABSTRACT FROM AUTHOR]

The binding capability of pyrrolidinyl peptide nucleic acids (PNAs) to DNA have been studied experimentally, also the high affinity and sequence specificity were reported. However, to date a detailed understanding of these properties is unclear due to a lack of crystallographic data. In this work, molecular dynamics (MD) simulations of pyrrolidinyl PNA with different stereochemistry, (2′ R ,4′ R )- and (2′ R ,4′ S )-prolyl-(1 S ,2 S )-2-aminocyclopentanecarboxylic acid (namely acpcPNA and epi -acpcPNA, respectively), binding to DNA were performed. MD simulations of three different forms (A-, B- and P-forms) were conducted to investigate the probable duplex conformation, which revealed that the MD structures were close to B-type duplex. It was found that acpcPNA and epi -acpcPNA backbones play a minor role in the hydrogen bond geometry of base pair, however, these greatly influence the interactions between PNA and DNA strands, leading to the differences in duplex stability. The calculated binding free energy revealed the higher thermodynamic stability of acpcPNA-DNA compared to epi -acpcPNA-DNA system, which is in good agreement with published experimental data. This work offers new insights into the conformational and energetic properties of pyrrolidinyl PNA-DNA duplexes at a molecular level. [ABSTRACT FROM AUTHOR]

DNA-based aptamers are widely employed as bioreceptors, owing to their tuneable affinity and specificity towards their targets. The use of peptide nucleic acids (PNAs) has instead proven challenging for this purpose, due to the absence of selection methods for the independent discovery of suitable receptors and due to the difficult mimicry of established DNA-based ones. Despite that PNAs exceed homologous DNA or RNA in terms of complementary base pairing, they can fail to reproduce alternative modes of binding because of their different structural features. The remarkable stability and charge distribution of PNAs could be beneficial to produce sensing bioreceptors, especially in the development of electronic devices such as field-effect transistor-based (FET) biosensors. We hereby report for the first time a high-affinity PNA aptamer for cardiac Troponin I (cTnI), a biomarker of acute myocardial infarction, able to interact with this specific protein in the picomolar range. The PNA aptamer was immobilized onto a graphene-based FET (gFET) transducer, and its ability in the direct detection of cTnI was compared with that of a DNA-based one of the same sequence. Similar dissociation constants were recorded for both receptors in 0.01 × PBS, as well as comparable detection limits of 6.0 ± 1.0 pg mL-1 (PNA aptamer) and 3.3 ± 0.7 pg mL-1 (DNA aptamer). Apart from the non-trivial demonstration that a PNA can behave as an aptamer, the tested receptor proved to be more consistent upon working in more complex biological matrices. [Display omitted] • First high-affinity PNA aptamer examplified for cardiac Troponin I (cTnI). • Integration of azide-terminated PNA onto graphene based field effect transistor. • PNA test receptor was highly adapted for working in more complex solutions. [ABSTRACT FROM AUTHOR]

The development of gold disk electrodes modified with single-stranded PNA (peptide nucleic acid) for the determination of important DNA sequences is described. The performance of anionic and cationic redox indicators for the generation of current signal is compared. It is shown that the use of cationic redox marker, namely hexaaminoruthenium(III) chloride results in a significantly better analytical parameters, as compared to measurements with other tested markers. The optimized sensor distinguishes itself with high selectivity toward complementary DNA sequences, with almost no electrochemical response toward DNA with one base mismatch. Developed PNA-based sensor was successfully used for detection of DNA sequence responsible for sickle cell anemia. [ABSTRACT FROM AUTHOR]

The development of gold disk electrodes modified with single-stranded PNA (peptide nucleic acid) for the determination of Hg2+ is described. PNA is the most useful and popular example of nucleic acid analogues. Due to the presence of glycine units linked via peptide bond instead of sugar-phosphate backbone, its interaction with molecules like proteins or metal ions is possible. The performance of anionic (AQMS) and cationic (MB and RuHex) redox markers for the generation of current signal is compared. It is shown that the use of anionic marker results in a significantly better analytical parameters, as compared to measurements with cationic markers. In the proposed system, the AQMS current difference increases proportionally within the Hg2+ concentration range 0.05 to 10 µmol⋅L-1, while for higher concentrations, the saturation of PNA layer with Hg2+ is observed. The optimized sensor distinguishes itself with high selectivity toward Hg2+ and lower detection limit of 3.33 nmol⋅L-1. The interactions between mercury ion and PNA strands were studied using electrochemical impedance spectroscopy (EIS) and spectrophotometry (UV-VIS). [ABSTRACT FROM AUTHOR]

Digital counting individual nucleic acid molecule is of great significance for fundamental biological research and accurate diagnosis of genetic diseases, which is hard to achieve with existing single-molecule detection technologies. Herein, we report a novel one-by-one single-molecule counting method for digital quantification of SARS-Cov-2 RNA. This method uses one fluorescent micromotor functionalized with peptide nucleic acids (PNAs) to specially capture one target RNA molecule. The RNA-micromotors can be propelled by the electric field to target district and accurately counted. Moreover, the method can also clearly discriminate one-base mutation in the target RNAs, indicating the great potential for clinical diagnostics and virus traceability survey. [Display omitted] • A one-by-one single RNA molecule counting method was firstly developed with nanomotors. • SARS-CoV-2 RNA variants could be identified accurately at single-nucleotide resolution. • Inspiring, the proposed approach should provide a powerful tool for counting all RNA/DNA targets. [ABSTRACT FROM AUTHOR]

Nanomaterial-based drug delivery has opened new horizons in cancer therapy. This study aimed to investigate the in vitro and in vivo anti-cancer effects of a hyaluronic acid (HA)-targeted nanocarrier based on hollow silica nanoparticles (HSNPs), gated with peptide nucleic acid (PNA) and ATP aptamer (ATP Apt) and loaded with doxorubicin (DOX). After formulation of a smart drug delivery nanosystem (HSNPs/DOX/ATP Apt /PNA/HA), drug release, cytotoxicity, uptake, and in vivo anti-tumor properties were studied. Drug release test showed the controlled release of encapsulated DOX in response to ATP content. MTT and flow cytometry indicated that HA could improve both cytotoxicity and cellular uptake of the formulation. Moreover, HA-targeted formulation enhanced both the survival rate and tumor inhibition in the tumor-bearing mice compared with free DOX (P < 0.05). Our findings confirmed that HA-targeted nanoformulation, gated with PNA/aptamer and loaded with DOX can provide a novel therapeutic platform with great potential for cancer therapy. In this study, hollow silica nanoparticles (HSNPs) were exploited to formulate a hyaluronic acid (HA)-targeted nanocarrier for delivery and controlled release of doxorubicin (DOX) to cancer cells. Peptide nucleic acid (PNA) and ATP aptamer (ATP Apt) were used as gating agents. HA-functionalized HSNPs/DOX/ATP Apt /PNA nanoplatforms internalize to cancer cells through HA-CD44 interaction. In the endosomes, higher ATP content triggers the dissociation of ATP Apt from its complementary strand on the HSNPs, forming an ATP Apt -ATP complex, eventually lead to release of DOX into the cytoplasm of cancer cells. [Display omitted] • Nanomaterials represent versatile applications in cancer treatment strategies. • Nanoparticle-based delivery is a revolutionary approach towards cancer treatment. • PNA-aptamer-gated hollow silica nanoparticles were targeted with hyaluronic acid. • Doxorubicin was encapsulated successfully in the constructed smart nanoparticles. • HSNPs/DOX/ATPApt/PNA/HA revealed considerable efficiency in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background: Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization. Results: Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe. Conclusions: The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums. [ABSTRACT FROM AUTHOR]

Background: Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization. Results: Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe. Conclusions: The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums. [ABSTRACT FROM AUTHOR]

An electrochemical DNA sensor based on ferrocene-labelled peptide nucleic acid (PNA-Fc) was prepared. The hybridization between PNA-Fc and DNA immobilized on a gold electrode was examined by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). PNA-Fc shows a good electrochemically activity and has a redox potential of 170 mV verus Ag/AgCl electrode after hybridization, representing the characteristic of ferrocene/ferrocenium (Fc/Fc+) transformation. The results illustrate that PNA-Fc can be used as an effective electrochemical DNA probe sensor. [ABSTRACT FROM AUTHOR]

• We developed a method for mitochondrial DNA SNP genotyping by melting curve analysis using PNA probes. • This method focuses on melting peak patterns obtained by thermal dissociation of PNA/DNA duplexes in amplified mtDNA products. • Compared to traditional sequencing, this method is more rapid and cost-effective. • SNP screening by this method may help minimize unnecessary recourse to full sequence analysis. • This method may be an effective and helpful tool in forensic investigations. Analysis of single nucleotide polymorphisms (SNPs) in mitochondrial (mt)DNA hypervariable regions (HV) 1/2 is valuable in forensic investigations. We developed a method for mtDNA screening of the HV1 and HV2 regions by melting curve analysis, using peptide nucleic acid (PNA) probes. This method focuses on melting peak patterns obtained by thermal dissociation of PNA/DNA duplexes in amplified mtDNA products. Five PNA probe sets were designed to detect 25 SNPs in the two HV regions. We also detected non-target SNPs based on unexpected melting temperature (T m) shifts. In fact, 62 SNPs (42 SNPs in HV1 and 20 in HV2) were identified, including the 25 target SNPs. Using this method, 46 melting peak patterns, including 8 pattern groups, were obtained in 60 unrelated individuals. The peak patterns were compared to 55 haplotypes identified by Sanger sequencing. The results obtained from analysis of target mtDNA SNPs were entirely consistent with those obtained by Sanger sequencing. Screening the HV1 and HV2 regions of mtDNA by this method may help minimize unnecessary recourse to full sequence analysis, allows to rapidly exclude samples that do not match evidence and reference samples, and may reduce turnaround times and analysis costs. Overall, this method may be effective and helpful in forensic investigations. [ABSTRACT FROM AUTHOR]

The article provides information on aspects related to use of CRISPR/Cas9 gene editing system and its potentiality to cure genetic disorders. Topics discussed include research findings for using synthetic peptide nucleic acid (PNA), production rate of oxygen carrying hemoglobin in human body, and correction of genetic error.