γ sulphate PNA (PNA S): Highly Selective DNA Binding Molecule Showing Promising Antigene Activity

Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described

Combination of the Systemin peptide with the beneficial fungus Trichoderma afroharzianum T22 improves plant defense responses against pests and diseases

Trichoderma spp. are among the most widely used plant beneficial fungi in agriculture. Its interaction with the plant triggers resistance responses by the activation of Induced Systemic Resistance mediated by Jasmonic acid and Ethylene and/or Systemic Acquired Resistance, which involves Salicylic acid, with the consequent control of a wide range of plant parasites. However, the benefit they can confer to plants may be reduced or nullified by environmental conditions or fungal ecological fitness. A novel approach to enhance their effectiveness in plant defense is to combine them with bioactive molecules including plant-derived compounds. Here, we show that plant treatment with Trichoderma afroharzianum (strain T22) and Systemin, a tomato peptide active in triggering plant defense, confers protection against the fungal pathogens Fusarium oxysporum, Botrytis cinerea and the insect pest Tuta absoluta. The observed defensive response was associated with an increase of Jasmonic acid and related metabolites and a decrease of Salicili acid

Liquid biopsy beyond cancer: a miRNA detection in serum with electrochemical chip for non-invasive coeliac disease diagnosis

Coeliac disease is a very common autoimmune disease estimated to affect 1 in 100 people worldwide. It occurs in genetically predisposed people where the ingestion of gluten leads to damage in the small intestine, and it is accurately diagnosticated through duodenal biopsy, an invasive diagnostic method. The liquid biopsy, generally used for monitoring cancer, is an appealing alternative even for autoimmune pathology such as coeliac disease, allowing for detecting disease progression or resistance to treatment. For this reason, an electrochemical peptide nucleic acid (PNA) device combined with a smartphone-assisted potentiostat for non-invasive coeliac disease diagnosis is proposed, by measuring the selected overexpressed miRNA-486-5p in serum, enlarging the application of liquid biopsy in nontumor pathologies. For highly sensitive detection, the polyester-based printed sensor is nanomodified with gold nanoparticles and a synthetic customized PNA probe. The designed sensor can detect the target analyte in the range of 10–100 nM with a limit of detection of 0.7 nM by measuring the variation of the response of the electrochemical mediator hexaammineruthenium in the presence of PNA–miRNA duplex on the nanostructured working electrode surface. The analyses testing serum samples are found in agreement with ones obtained by realxtime quantitative polymerase chain reaction (RT-qPCR), demonstrating the reliability of this innovative electrochemical chip developed

Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

Ultraconserved regions (UCRs) have been shown to originate non-coding RNA transcripts (T-UCRs) that have different expression profiles and play functional roles in the pathophysiology of multiple cancers. The relevance of these functions to the pathogenesis of bladder cancer (BlCa) is speculative. To elucidate this relevance, we first used genome-wide profiling to evaluate the expression of T-UCRs in BlCa tissues. Analysis of two datasets comprising normal bladder tissues and BlCa specimens with a custom T-UCR microarray identified ultraconserved RNA (uc.) 8+ as the most upregulated T-UCR in BlCa tissues, although its expression was lower than in pericancerous bladder tissues. These results were confirmed on BlCa tissues by real-time PCR and by in situ hybridization. Although uc.8+ is located within intron 1 of CASZ1, a zinc-finger transcription factor, the transcribed non-coding RNA encoding uc.8+ is expressed independently of CASZ1. In vitro experiments evaluating the effects of uc.8+ silencing, showed significantly decreased capacities for cancer cell invasion, migration, and proliferation. From this, we proposed and validated a model of interaction in which uc.8+ shuttles from the nucleus to the cytoplasm of BlCa cells, interacts with microRNA (miR)-596, and cooperates in the promotion and development of BlCa. Using computational analysis, we investigated the miR-binding domain accessibility, as determined by base-pairing interactions within the uc.8+ predicted secondary structure, RNA binding affinity, and RNA species abundance in bladder tissues and showed that uc.8+ is a natural decoy for miR-596. Thus uc.8+ upregulation results in increased expression of MMP9, increasing the invasive potential of BlCa cells. These interactions between evolutionarily conserved regions of DNA suggest that natural selection has preserved this potentially regulatory layer that uses RNA to modulate miR levels, opening up the possibility for development of useful markers for early diagnosis and prognosis as well as for development of new RNA-based cancer therapies

Synthesis and characterization of PNA based molecules able to interfere with gene regulation

The control of gene expression is a fundamental process to bring the genome to life, and it pervades most of biology, from cell proliferation and differentiation to development. Mis-regulation of gene expression at any level can lead to disease.MicroRNAs, single-stranded RNAs (ssRNAs) of 19–25 nu-cleotides in length,function as guide molecules in post-transcriptional gene silencing by base pairing with target mRNAs, which leads to mRNA cleavage or translational repression. In this work PNA based molecules designed to interfere in the maturation of miRNA and gene expression were obtained.To achieve these goals, firstly a new, efficient and inexpensive protocol to obtain PNA by Fmoc solid-phase synthesis was developed. To interfere in the miRNA210 function PNA5 and PNA6 anti-premiR were obtained.To improve PNA delivery in cells, conjugation to cell-penetrating peptides, such as Tat peptide, and nuclear localization signal (NLS and biNLS) peptides was carried out. Furthermore, in order to demonstrate that the designed PNAs were able to bind to the pre-miRNA210 a Thiazole Orange (TO) modified PNA for fluorescence studies was obtained.A further aim of this project was the synthesis and the characterization of new modified PNA monomers bearing a sulphate group in gamma position of the backbone and of oligomers containing sulphate monomers were set up. The conformational preferences of the PNA monomers were investigated by NMR. Studies on the secondary structure of a polypirimidine oligomer were carried out by CD. The ability of the modified oligomer to interact with DNA, the specificity and affinity of binding were investigated by UV and CD. Finally, the ability of the sulphate PNA to interfere with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), by FACS and qPCR was explored

Interactions of antimicrobial peptides with bacterial membrane model systems and with whole E. coli bacterial cells

Antimicrobial peptides are a primitive component of the innate immune system, produced by organisms after the invasion of pathogens. Extensive studies have led to the discovery of a wide variety of such peptides, from diverse organisms, showing different activities and structures. Studies on peptides secreted on the frog skin allowed the characterization of a class of short peptides, temporins, characterized by strong antimicrobial activity against Gram-positive bacteria and fungi. We have recently developed a Temporin B analogue, named TB_G6AKK, composed of natural amino acids, showing activity at low concentrations either against Gram-positive and Gram-negative bacteria (Avitabile et al 2013, in press). TB_G6AKK interacts with E.coli LPS, as demonstrated by fluorescence, CD and ITC studies, and folds as a kinked helix upon interaction with the E.coli LPS, as demonstrated by NMR. Studies aimed to characterize the ability of this peptide to kill pathogens as Pseudomonas aeruginosa, whose infection is cause of complications in patients suffering cystic fibrosis are currently in progress. In order to deeply characterize the interactions of such peptide with the bacterial cells, as LPS is only one of the components of the bacterial cell wall, we started to explore the secondary structure of the peptide in the presence of whole bacterial cells by CD and by fluorescence the binding of the peptide to the cells. Analysis of the data obtained with cells and comparison with those obtained with LPS will help us to understand the effectiveness of the LPS as a model system to represent the cells Gram negative bacteria

Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed

Inhibition of miRNA maturation by peptide nucleic acids

Molecules able to interfere in miRNA genesis and function are potent tools to unravel maturation and processing pathways. Antisense oligonucleotides or analogs are actually employed for the inhibition of miRNA function. Here we illustrate how Peptide Nucleic Acids oligomers targeting pre-miRNA are exploited to inhibit miRNA maturation

The influence of liposomal formulation on the incorporation and retention of PNA oligomers

Liposomal formulations composed of phospholipids with different unsaturation degrees, head groupsand at different cholesterol content have been tested for the encapsulation of Peptide Nucleic Acid (PNA)oligomers. The best loading capability (177 g, ER% = 87.2) was obtained for pure liposomes of phos-phatidylglycerol (DOPG) with negatively charged head group. The insertion of a 10–20% of cholesterol inDOPG based liposomes provides a slight decrease (∼160 g) of the PNA loading. On the other hand, thecholesterol addition (20–30%) slows down the PNA’s release (∼27%) in fetal bovine serum from the lipo-somal formulation. Based on the encapsulation and the release properties, PEGylated DOPG liposomeswith a percentage of cholesterol of 10–20% are the optimal formulation for the loading of PNA-a210