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Abstract: Technologies today available for the DNA detection rely on a combination of labeled probes hybridized to target sequences which are amplified by polymerase chain reaction (PCR). Direct detection methods that eliminate the requirement for both PCR and labeling steps could afford faster, cheaper and simpler devices for the analysis of small amounts of unamplified DNA. In this work we describe the results obtained in the ultrasensitive detection of non-amplified genomic DNA. We analyzed certified reference materials containing different amounts of genetically modified DNA by using a detection method which combines the nanoparticle-enhanced surface plasmon resonance imaging (SPRI) biosensing to the peptide nucleic acids (PNAs) improved selectivity and sensitivity in targeting complementary DNA sequences. The method allowed us to obtain a 41zM sensitivity in targeting genomic DNA even in the presence of a large excess of non-complementary DNA. [Copyright &y& Elsevier]

We have shown previously that a T10 peptide nucleic acid (PNA) bound to the transcriptional terminator of a Saccharomyces cerevisiae tDNAIle(TAT) gene arrests elongating yeast RNA polymerase (pal) III at a position that precedes by 20 bp the upstream end of the PNA roadblock (Dieci, G., Corradini, R., Sforza, S., Marchelli, R., and Ottonello, S. (2001) J. Biol. Chem. 276, 5720–5725). Here, a PNA-binding cassette was placed at various distances downstream of a functional tDNAIle transcriptional terminator (T6) that is not bound by the T10 PNA, and the effect of the PNA roadblock on RNA 3′-end formation, transcript release, and transcription reinitiation was examined. With a PNA roadblock placed as close as 5 hp downstream of the T6 terminator, pol III could still reach the termination site and complete pre-tRNA synthesis, implying that the catalytic site-to-front edge (C-F) distance of the polymerase can shorten by >10 bp upon recognition of the terminator element. In addition, transcripts synthesized by a PNA-roadblocked terminating pal III were found to be released from transcription complexes. Interestingly, however, the same roadblock dramatically reduced the rate of transcription reinitiation. Also, when placed 5 bp downstream of a mutationally inactivated terminator element (T3GT2), the PNA roadblock restored transcription termination, thus indicating that the inactivated terminator is compromised in its ability to cause pol III pausing, but can still induce C-F distance shortening and transcript release. The latter two activities were found to be further impaired in variants of the inactivated terminator bearing fewer than three consecutive T residues (T2G2T2 and TG2TGT). The data indicate that RNA polymerase pausing, C-F distance shortening, and transcript release are functionally distinguishable features of the termination process and point to the RNA release propensity of pol III as a major determinant of its remarkably high termination efficiency. [ABSTRACT FROM AUTHOR]

Chiral peptide nucleic acid (PNA) monomers based on the amino acids Ala, Phe and Lys were synthesized, and their enantiomeric purity was checked either by RP-HPLC after reaction with l-ValOMe or by GC–MS using a Chirasil-Val column after hydrolysis and conversion of PNAs to the corresponding piperazin-2-ones. A model coupling reaction of these monomers with ValOMe was carried out under various conditions in order to evaluate the effect of synthetic parameters (coupling agent, base, preactivation time) on epimerization. The enantiomeric purity of the products decreased in the order: DEPBT>TDBTU>HBTU>HATU. The use of sym-collidine (TMP) as a base produced higher racemization than with DIEA. PNAs containing one or three chiral monomers were subsequently synthesized with different coupling protocols, and the results were found to be consistent with those obtained in solution. High enantiomeric purity was obtained using a DIC/HOBt coupling protocol. A rationale for the observed effects is proposed based on NMR studies. [Copyright &y& Elsevier]

Uses the copper(II) complex of a histamine-modified cyclodextrin (6-deoxy-6-N-histamine-beta-cyclodextrin, CDhm) for the chiral recognition of amino acids. HPLC separation of the enantiomers of unmodified aromatic amino acids; CD spectra of complexes containing aromatic amino acids.

Focuses on the metal binding properties of cyclodextrins, used in building blocks for supramolecular structures, and the modification of cyclodextrins to achieve a more efficient binding of molecules with complementary functionalities. How to achieve modification; Metal binding properties of cyclodextrins.

The article discusses peptide nucleic acid (PNA) and its scientific application and molecular structure. Scientific research of PNA in various fields of science are discussed including DNA nanotechnology, molecular biology, and medicine. Articles in the issue regarding medicinal plants, and gene editing techniques are also discussed.

An introduction is presented in which the editors discusses a series of papers honoring Professor Rosangela Marchelli with works ranging from organic synthesis to biomedical applications of peptide nucleic acids (PNA).

One of the most appealing approaches for regulating gene expression, named the "microRNA therapeutic" method, is based on the regulation of the activity of microRNAs (miRNAs), the intracellular levels of which are dysregulated in many diseases, including cancer. This can be achieved by miRNA inhibition with antimiRNA molecules in the case of overexpressed microRNAs, or by using miRNA-mimics to restore downregulated microRNAs that are associated with the target disease. The development of new efficient, low-toxic, and targeted vectors of such molecules represents a key topic in the field of the pharmacological modulation of microRNAs. We compared the delivery efficiency of a small library of cationic calix[4]arene vectors complexed with fluorescent antimiRNA molecules (Peptide Nucleic Acids, PNAs), pre-miRNA (microRNA precursors), and mature microRNAs, in glioma- and colon-cancer cellular models. The transfection was assayed by cytofluorimetry, cell imaging assays, and RT-qPCR. The calix[4]arene-based vectors were shown to be powerful tools to facilitate the uptake of both neutral (PNAs) and negatively charged (pre-miRNAs and mature microRNAs) molecules showing low toxicity in transfected cells and ability to compete with commercially available vectors in terms of delivery efficiency. These results could be of great interest to validate microRNA therapeutics approaches for future application in personalized treatment and precision medicine. [ABSTRACT FROM AUTHOR]

Modification of the PNA backbone can be used to broaden their utility by introducing new functional groups. In particular, gamma-modified PNA have been found to be quite effective in a number of applications, and exhibit particularly high DNA binding affinity. The introduction of one side chain imply that the achiral backbone of PNA becomes chiral, and binding properties depend on the stereochemistry. A new paper on gamma-modified PNA by Ly and co-workers complete the existing knowledge by displaying that in binding to complementary PNA stereochemical orthogonality can be demonstrated. This opens the way to the exploitation of stereochemical features in diagnostic assays and in nanofabrication. [ABSTRACT FROM PUBLISHER]

Simple Summary: Starting from a list of nine miRNAs found to be dysregulated in the bloodstream of early-stage colon cancer (CRC) patients, the biological effects of the targeting of the most relevant up-regulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified. The down-regulation of these target miRNAs with PNAs is associated with apoptosis induction in CRC cellular models. Apoptosis induction, which is one of the most important anticancer mechanisms, reaches very high levels when anti-miRNAs PNAs are combined with other pro-apoptotic compounds such as sulforaphane. Liquid biopsy has dramatically changed cancer management in the last decade; however, despite the huge number of miRNA signatures available for diagnostic or prognostic purposes, it is still unclear if dysregulated miRNAs in the bloodstream could be used to develop miRNA-based therapeutic approaches. In one author's previous work, nine miRNAs were found to be dysregulated in early-stage colon cancer (CRC) patients by NGS analysis followed by RT-dd-PCR validation. In the present study, the biological effects of the targeting of the most relevant dysregulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified, and their anticancer activity in terms of apoptosis induction was evaluated. Our data demonstrate that targeting bloodstream up-regulated miRNAs using anti-miRNA PNAs leads to the down-regulation of target miRNAs associated with inhibition of the activation of the pro-apoptotic pathway in CRC cellular models. Moreover, very high percentages of apoptotic cells were found when the anti-miRNA PNAs were associated with other pro-apoptotic agents, such as sulforaphane (SFN). The presented data sustain the idea that the targeting of miRNAs up-regulated in the bloodstream with a known role in tumor pathology might be a tool for the design of protocols for anti-tumor therapy based on miRNA-targeting molecules. [ABSTRACT FROM AUTHOR]

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene encodes for a chloride channel defective in Cystic Fibrosis (CF). Accordingly, upregulation of its expression might be relevant for the development of therapeutic protocols for CF. MicroRNAs are deeply involved in the CFTR regulation and their targeting with miRNA inhibitors (including those based on Peptide Nucleic Acids, PNAs)is associated with CFTR upregulation. Targeting of miR-145-5p, miR-101-3p, and miR-335-5p with antisense PNAs was found to be associated with CFTR upregulation. The main objective of this study was to verify whether combined treatments with the most active PNAs are associated with increased CFTR gene expression. The data obtained demonstrate that synergism of upregulation of CFTR production can be obtained by combined treatments of Calu-3 cells with antisense PNAs targeting CFTR-regulating microRNAs. In particular, highly effective combinations were found with PNAs targeting miR-145-5p and miR-101-3p. Content of mRNAs was analyzed by RT-qPCR, the CFTR production by Western blotting. Combined treatment with antagomiRNAs might lead to maximized upregulation of CFTR and should be considered in the development of protocols for CFTR activation in pathological conditions in which CFTR gene expression is lacking, such as Cystic Fibrosis. [ABSTRACT FROM AUTHOR]

In this paper, a novel platform for lab-in-fiber-based biosensors is studied. Hollow-core tube lattice fibers (HC-TLFs) are proposed as a label-free biosensor for the detection of DNA molecules. The particular light-guiding mechanism makes them a highly sensitive tool. Their transmission spectrum is featured by alternations of high and low transmittance at wavelength regions whose values depend on the thickness of the microstructured web composing the cladding around the hollow core. In order to achieve DNA detection by using these fibers, an internal chemical functionalization process of the fiber has been performed in five steps in order to link specific peptide nucleic acid (PNA) probes, then the functionalized fiber was used for a three-step assay. When a solution containing a particular DNA sequence is made to flow through the HC of the TLF in an 'optofluidic' format, a bio-layer is formed on the cladding surfaces causing a red-shift of the fiber transmission spectrum. By comparing the fiber transmission spectra before and after the flowing it is possible to identify the eventual formation of the layer and, therefore, the presence or not of a particular DNA sequence in the solution. [ABSTRACT FROM AUTHOR]

Abstract: N γ-Carboxyalkyl modified peptide nucleic acids (PNAs), containing the four canonical nucleobases, were prepared via solid-phase oligomerization. The inserted peptoid monomers 1 and 2 were constructed through simple synthetic procedures, utilizing appropriate glycidol and iodoalkyl electrophiles. Thermal denaturation studies, performed with complementary antiparallel DNA strands, demonstrated that the length of the N γ-side chain strongly influences the modified PNAs hybridization properties. Moreover, multiple negative charges on the oligoamide backbone, when present on γ-nitrogen C6 side chains proved to be beneficial for the oligomers’ water solubility and DNA hybridization specificity. [Copyright &y& Elsevier]

Abstract: Lys-based ‘chiral box’ Peptide Nucleic Acids (PNAs with three adjacent 2D-Lys-based chiral monomers) have shown unsurpassed specificity in DNA recognition. In this Letter, the binding performances of arginine-based chiral PNAs were evaluated for PNAs containing in the middle part of the strand either a 2D,5L-Arg monomer or three adjacent 2D-; 2D,5L-; 5L-Arg monomers (‘Extended Chiral Box’), a combination never studied before. The binding performances of the PNAs were studied by evaluating the melting temperatures of fullmatch and mismatch PNA–DNA and PNA–RNA hybrids and by studying their structure by circular dichroism (CD). The data indicated that the arginine side chains inserted in the PNA structure are perfectly equivalent to lysine side chains as far as oligonucleotide recognition is concerned. The insertion of an ‘Extended Chiral Box’ into PNA differently influences the binding properties to DNA and RNA: the additional side chains had no observable effect on binding affinity and selectivity toward DNA, whereas, seemed to slightly disturb the binding affinity to RNA but at the same time highly enhancing the recognition selectivity. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of new chiral PNA analogues based on lysine is reported. In particular, l- and/or d-lysine-based PNA submonomers bearing two lysine side chains exactly spaced as in the dipeptide Lys-Lys were synthesized and incorporated in the middle of decameric PNA strands, obtaining four diastereomeric (LD, DL, LL and DD) lysine-based chiral PNAs. The hybridization with their complementary antiparallel DNA strand was studied by melting temperature determination and compared with the analogue achiral PNA and chiral PNAs bearing one residue with either of the two lysine enantiomers. The binding abilities were shown to be strongly dependent on the configuration of the stereogenic centres. [Copyright &y& Elsevier]

The study of unsubstituted and disubstituted β-cyclodextrins (β-CDs) by ESI-mass spectrometry is reported, applying a cone-induced fragmentation in the presence of a twofold excess of sodium chloride, in order to gain information about the fragmentation of the different regioisomers. On the basis of the fragmentation pattern observed for the unsusbstituted β-CD, a statistical model shows that the fragments generated by every regioisomer of a disubstituted CD (AB, AC, and AD) are expected to differ in their relative intensity and, therefore, they can be used for correctly identifying the three different regioisomers. The model was tested on the three regioisomeric (AB, AC, and AD) diamino-β-CDs and ditosyl-β-CD and on the AC and AD regioisomers of dimesitylenesulphonyl-β-CD, allowing in every case through statistical analysis of the fragmentation pattern the correct assignment of every regioisomer on the basis of an ESI mass spectrum (single quadrupole analyzer, high cone voltage) of the pure compounds. The absolute intensities of the fragmentation peaks were voltage-dependent but their ratios was voltage-independent, indicating that no mass bias in peak ratios is introduced by the analyzer. Given the fast time of analysis and its general applicability, independently from the substituents, we propose our method as an easy way to identify the regioisomers of disubstituted β-CDs. [Copyright &y& Elsevier]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract A novel amperometric genosensor based on PNA probes covalently bound on the surface of Single Walled Carbon Nanotubes – Screen Printed Electrodes (SWCNT-SPEs) was developed and validated in samples of non-amplified genomic DNA extracted from genetically modified (GM)-Soy. The sandwich assay is based on a first recognition of a 20-mer portion of the target DNA by a complementary PNA Capture Probe (CP) and a second hybridization with a PNA Signalling Probe (SP), with a complementary sequence to a different portion of the target DNA. The SP was labelled with biotin to measure current signal by means of a final incubation of an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). The electrochemical detection was carried out using hydroquinone diphosphate (HQDP) as enzymatic substrate. The genoassay provided a linear range from 250 pM to 2.5 nM, LOD of 64 pM and LOQ of 215 pM Excellent selectivity towards one base mismatch (1-MM) or scrambled (SCR) sequences was obtained. A simple protocol for extraction and analysis of non-amplified soybean genomic DNA without sample treatment was developed and validated. Our study provides insight into how the outstanding recognition efficiency of PNAs can be combined with the unique properties of CNTs in terms of signal response enhancement for direct detection of genomic DNA samples at the level of interest without previous amplification. Highlights • First amperometric genosensor based on PNA immobilized on carbon nanotubes. • PNA provided high selectivity while carbon nanotubes exhibited high sensitivity. • Direct determination of genomic DNA from GM Soy, without preamplification. • Validation on soybean flour, matching the EC Regulation (0.9% threshold limit). • Screening tool valuable for food authenticity assessment. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level. [ABSTRACT FROM AUTHOR]

Over the past decades, peptide nucleic acid/DNA (PNA:DNA) duplex stability has been improved via backbone modification, often achieved via introducing an amino acid side chain at the α- or γ-position in the PNA sequence. It was previously shown that interstrand cross-linking can further enhance the binding event. In this work, we combined both strategies to fine-tune PNA crosslinking towards single stranded DNA sequences using a furan oxidation-based crosslinking method; for this purpose, γ-L-lysine and γ-L-arginine furan-PNA monomers were synthesized and incorporated in PNA sequences via solid phase synthesis. It was shown that the L-lysine γ-modification had a beneficial effect on crosslink efficiency due to pre-organization of the PNA helix and a favorable electrostatic interaction between the positively-charged lysine and the negatively-charged DNA backbone. Moreover, the crosslink yield could be optimized by carefully choosing the type of furan PNA monomer. This work is the first to describe a selective and biocompatible furan crosslinking strategy for crosslinking of γ-modified PNA sequences towards single-stranded DNA. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are polyamide analogues of nucleic acids, very effective in terms of affinity and selectivity in DNA/RNA recognition. As other supramolecular entities, the PNA structure has been an interesting scaffold for the development of new molecules, aimed to DNA and RNA recognition, with improved or completely new properties. This review describes recent work, with the aim of describing how the design of these molecules has evolved in recent years, using increasingly effective tools, from simple crystal structure analysis to molecular dynamics and metadynamics. Modified PNA with additional modules appended, either on the backbone or on the nucleobase, are described. Polyfunctional molecules with both backbone and nucleobase modification are then considered. Finally, recent examples of architectures obtained by conjugation of PNAs to inorganic nanostructures as cargo systems for diagnostics and nano-biotechnology are presented. [ABSTRACT FROM AUTHOR]

A DNA-sensing platform is developed by exploiting the easy surface functionalization of metal-organic framework (MOF) particles and their highly parallelized fluorescence detection by flow cytometry. Two strategies were employed to functionalize the surface of MIL-88A, using either covalent or non-covalent interactions, resulting in alkyne-modified and biotin-modified MIL-88A, respectively. Covalent surface coupling of an azide-dye and the alkyne-MIL-88A was achieved by means of a click reaction. Non-covalent streptavidin-biotin interactions were employed to link biotin-PNA to biotin-MIL-88A particles mediated by streptavidin. Characterization by confocal imaging and flow cytometry demonstrated that DNA can be bound selectively to the MOF surface. Flow cytometry provided quantitative data of the interaction with DNA. Making use of the large numbers of particles that can be simultaneously processed by flow cytometry, this MOF platform was able to discriminate between fully complementary, single-base mismatched, and randomized DNA targets. [ABSTRACT FROM AUTHOR]

The direct delivery of specific proteins to live cells promises a tremendous impact for biological and medical applications, from therapeutics to genetic engineering. However, the process mostly involves tedious techniques and often requires extensive alteration of the protein itself. Herein we report a straightforward approach to encapsulate native proteins by using breakable organosilica matrices that disintegrate upon exposure to a chemical stimulus. The biomolecule-containing capsules were tested for the intracellular delivery of highly cytotoxic proteins into C6 glioma cells. We demonstrate that the shell is broken, the release of the active proteins occurs, and therefore our hybrid architecture is a promising strategy to deliver fragile biomacromolecules into living organisms. [ABSTRACT FROM AUTHOR]

The direct delivery of specific proteins to live cells promises a tremendous impact for biological and medical applications, from therapeutics to genetic engineering. However, the process mostly involves tedious techniques and often requires extensive alteration of the protein itself. Herein we report a straightforward approach to encapsulate native proteins by using breakable organosilica matrices that disintegrate upon exposure to a chemical stimulus. The biomolecule-containing capsules were tested for the intracellular delivery of highly cytotoxic proteins into C6 glioma cells. We demonstrate that the shell is broken, the release of the active proteins occurs, and therefore our hybrid architecture is a promising strategy to deliver fragile biomacromolecules into living organisms. [ABSTRACT FROM AUTHOR]

Parallel PNA:PNA duplexes were synthesized and conjugated with meso-tris(pyridyl)phenylporphyrin carboxylic acid at the N-terminus. The introduction of one porphyrin unit was shown to affect slightly the stability of the PNA:PNA parallel duplex, whereas the presence of two porphyrin units at the same end resulted in a dramatic increase of the melting temperature, accompanied by hysteresis between melting and cooling curves. The circular dichroism (CD) profile of the Soret band and fluorescence quenching strongly support the occurrence of a face-to-face interaction between the two porphyrin units. Introduction of a L-lysine residue at the C-terminal of one strand of the parallel duplex induced a left-handed helical structure in the PNA:PNA duplex if the latter contains only one or no porphyrin moiety. The left-handed helicity was revealed by nucleobase CD profile at 240-280 nm and by the induced-CD observed in the presence of the DiSC2(5) cyanine dye at ~500-550 nm. Surprisingly, the presence of two porphyrin units led to the disappearance of the nucleobase CD signal and the absence of CD exciton coupling within the Soret band region. In addition, a dramatic decrease of induced CD of DiSC2(5) was observed. These results are in agreement with a model where the porphyrin-porphyrin interactions cause partial loss of chirality of the PNA:PNA parallel duplex, forcing it to adopt a ladder-like conformation. Chirality 27:864-874, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Microstructured optical fibers containing microchannels and Bragg grating inscribed were internally functionalized with a peptide nucleic acid (PNA) probe specific for a gene tract of the genetically modified Roundup Ready soy. These fibers were used as an optofluidic device for the detection of DNA by measuring the shift in the wavelength of the reflected IR light. Enhancement of optical read-out was obtained using streptavidin coated gold-nanoparticles interacting with the genomic DNA captured in the fiber channels (0%, 0.1%, 1% and 10% RR-Soy), enabling to achieve statistically significant, label-free, and amplification-free detection of target DNA in low concentrations, low percentages, and very low sample volumes. Computer simulations of the fiber optics based on the finite element method (FEM) were consistent with the formation of a layer of organic material with an average thickness of 39 nm for the highest percentage (10% RR soy) analysed. [ABSTRACT FROM AUTHOR]

The design and synthesis of multifunctional nanomaterials could lead to applications relevant for biomedicine. Manufacturing porous particles to make them able to carry bioactive molecules into living cells represents a substantial goal towards the development of powerful tools for nanomedicine. This work describes a first example of using zeolite-L crystals as multifunctional nanocontainers to simultaneously deliver DNA oligonucleotides and organic molecules into living cells. Multifunctional zeolite-L was prepared by filling the pore system with guest molecules, whilst DNA was adsorbed electrostatically on their surface. The release kinetics of DNA and of the guest molecules into living cells was studied to prove the multiple-drug-delivery ability of the system. The localization of all the components in different cellular compartments was followed. The presented system may be a prototype for the development of novel nanoparticles for drug delivery and gene therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We describe a novel sensing approach based on a functionalized microstructured optical fiber-Bragg grating for specific DNA target sequences detection. The inner surface of a microstructured fiber, where a Bragg grating was previously inscribed, has been functionalized by covalent linking of a peptide nucleic acid probe targeting a DNA sequence bearing a single point mutation implicated in cystic fibrosis (CF) disease. A solution of an oligonucleotide (ON) corresponding to a tract of the CF gene containing the mutated DNA has been infiltrated inside the fiber capillaries and allowed to hybridize to the fiber surface according to the Watson-Crick pairing. In order to achieve signal amplification, ON-functionalized gold nanoparticles were then infiltrated and used in a sandwich-like assay. Experimental measurements show a clear shift of the reflected high order mode of a Bragg grating for a 100 nMDNA solution, and fluorescence measurements have confirmed the successful hybridization. Several experiments have been carried out on the same fiber using the identical concentration, showing the same modulation trend, suggesting the possibility of the reuse of the sensor. Measurements have also been made using a 100 nM mismatched DNA solution, containing a single nucleotide mutation and corresponding to the wild-type gene, and the results demonstrate the high selectivity of the sensor. [ABSTRACT FROM AUTHOR]

The use of droplet-based microfluidics and peptide nucleic acid molecular beacons for the detection of polymerase chain reaction (PCR)-amplified DNA sequences within nanoliter-sized droplets is described in this work. The nanomolar-attomolar detection capabilities of the method were preliminarily tested by targeting two different single-stranded DNA sequences from the genetically modified Roundup Ready soybean and the Olea europaea genomes and detecting the fluorescence generated by peptide nucleic acid molecular beacons with fluorescence microscopy. Furthermore, the detection of 10 nM solutions of PCR amplicon of DNA extracted from leaves of O. europaea L. encapsulated in nanoliter-sized droplets was performed to demonstrate that peptide nucleic acid molecular beacons can discriminate O. europaea L. cultivar species carrying different single-nucleotide polymorphisms. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition. [ABSTRACT FROM AUTHOR]

PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut ( ) oil in extra-virgin olive oil ( L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. [ABSTRACT FROM AUTHOR]

Abstract: The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects. [Copyright &y& Elsevier]

Abstract: The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo. [Copyright &y& Elsevier]

Abstract: A new submonomeric approach for the Fmoc solid phase synthesis of chiral PNAs is reported here. The design and synthesis of a new d-lysine-based Fmoc submonomer obtained by replacing the nucleobase residue with an Alloc group, compatible with Fmoc chemistry, is described starting from d-lysine and Fmoc-aminoacetaldehyde. The desired submonomer was obtained in high yield with no racemization. The conditions for synthesizing a chiral PNA dimer on solid support by a submonomeric approach have been optimized, allowing to obtain the desired PNA with a very high optical purity. [Copyright &y& Elsevier]

Abstract: The 3,3′-diethylthiacarbocyanine (DiSC2(5)) dye is able to aggregate on full matched PNA–DNA duplexes by changing its absorption properties, which are manifested by an instantaneous colour shift from blue to purple. However the spontaneous aggregation of the dye also on mismatched duplexes and even on free PNA strands makes the test quite aspecific. Here it is demonstrated that the addition of succinyl-β-cyclodextrin (Succ-β-CyD) to the solutions containing PNA–DNA duplexes and the dye strongly enhances the specificity of the colour shift, allowing for a fast, very specific and extremely sensitive visual recognition of mismatches in DNA strands by using PNA probes in combination with the DiSC2(5) dye. The phenomenon has been studied by CD and NMR spectroscopies. The method has been optimized and preliminarily applied for the recognition of an apoE gene mutation in human DNA samples. [Copyright &y& Elsevier]

The conjugation of a bioactive, fluorescent PNA sequence to high-molecular weight poly(ethylene glycol) (PEG) is described and the properties of the PEG-PNA conjugate are evaluated. [ABSTRACT FROM AUTHOR]

Calcein-acetoxymethylester (calcein-AM) is a non-fluorescent, cell permeant compound, which is converted by intracellular esterases into calcein, an anionic fluorescent form. It is used in microscopy and fluorometry and provides both morphological and functional in- formation of viable cells. In this study we have tested the response of calcein-AM to oxidation. In cell-free fluorometric assays, H2O2 and xanthine-xanthine oxidase induced a dose-dependent emission of the AM form but had no effects on calcein. Fluorometric and confocal microscopy tests on human fibroblasts confirmed that the cell permeant AM form is the actual sensor since its removal from culture medium, and its consequent back-diffusion, made the system insensitive to oxidative stimuli. In time- lapse confocal microscopy, calcein-AM detected changes in the intracellular redox state following direct oxidation (H2O2, xanthine-xanthine oxidase) and phorbol ester treatment. Comparative tests showed that calcein-AM sensitivity to oxidation is about one order of magnitude higher than other fluorescein derivatives. The absence of leakage, due to the presence of the probe in the extracellular compartment, and its low toxicity allow to perform experiments for prolonged times following the response to the same or different stimuli repeatedly applied. We propose calcein-AM as a sensitive tool for intracellular ROS generation in living cells with useful applications for real-time imaging in confocal microscopy. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as D-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three D-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a D-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-A resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The D-Lys-based chiral PNA-DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition. [ABSTRACT FROM AUTHOR]

A novel enzyme-labelled voltammetric magnetogenoassay for DNA sensing based on the use of carboxyl-surface coated magnetic microbeads functionalized with PNA probes and subsequent read-out on screen-printed electrode (SPE) substrates was developed. The assay was validated for determination of non-amplified genomic DNA from genetically modified Roundup Ready soy. Outstanding performance with respect to other genoassays requiring preliminary amplification of target DNA via PCR was demonstrated. The analytical performance was also improved compared to previous methods based on the immobilization of the same PNA probes on SPE substrates, since the method was found capable of achieving LOD and LOQ of 415 fM and 995 fM, respectively. The ability of the magnetogenoassay to detect the presence of Roundup Ready soy DNA sequence was tested on genomic DNA extract from European Reference Material soy flours, demonstrating the capability of the method to match the European Union regulation for labelling of food containing a percentage of GM products greater than 0,9%. [Display omitted] • A sandwich electrochemical magnetogenoassay for the determination of DNA from GM soy was developed. • Fitness for application performed on soy flours reaching femtomolar LOD. • Valuable analytical performances for food safety and authenticity assessment. [ABSTRACT FROM AUTHOR]

Since the identification of microRNAs (miRNAs) involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, miRNAs known to down-regulate the expression of the CFTR and associated proteins have been investigated as potential therapeutic targets. Here we show that miR-101-3p, targeting the 3′-UTR sequence of the CFTR mRNA, can be selectively inhibited by a peptide nucleic acid (PNA) carrying a full complementary sequence. With respect to clinical relevance of microRNA targeting, it is expected that reduction in concentration of miRNAs (the anti-miRNA approach) could be associated with increasing amounts of target mRNAs. Consistently to this hypothesis, we report that PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. Next Generation Sequencing (NGS) was performed in order to verify the effects of the anti-miR-101-3p PNA on the Calu-3 miRNome. Upon inhibition of miR-101-3p we observed a fold change (FC) expression <2 of the majority of miRNAs (403/479, 84.13%), whereas we identified a list of dysregulated miRNAs, suggesting that specific miRNA inhibition (in our case miR-101-3p) might be accompanied by alteration of expression of other miRNAs, some of them known to be involved in Cystic Fibrosis (CF), such as miR-155-5p and miR-125b-5p. Image 1 • microRNAs are deeply involved in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. • miR-101-3p targets the 3′-UTR sequence of the CFTR mRNA. • In Calu-3 cells miR-101-3p can be selectively inhibited by a fully complementary peptide nucleic acid (PNA). • PNA-mediated inhibition of miR-101-3p was accompanied by CFTR up-regulation. • The effects of the anti-miR-101-3p PNA on the Calu-3 miRNome are accompanied by alteration of miRNAs involved in Cystic Fibrosis. [ABSTRACT FROM AUTHOR]

The number of applications of peptide nucleic acids (PNAs)—oligonucleotide analogs with a polyamide backbone—is continuously increasing in both in vitro and cellular systems and, parallel to this, delivery systems able to bring PNAs to their targets have been developed. This review is intended to give to the readers an overview on the available carriers for these oligonucleotide mimics, with a particular emphasis on newly developed multi-component- and multifunctional vehicles which boosted PNA research in recent years. The following approaches will be discussed: (a) conjugation with carrier molecules and peptides; (b) liposome formulations; (c) polymer nanoparticles; (d) inorganic porous nanoparticles; (e) carbon based nanocarriers; and (f) self-assembled and supramolecular systems. New therapeutic strategies enabled by the combination of PNA and proper delivery systems are discussed. [ABSTRACT FROM AUTHOR]

RAS mutations in the blood of colorectal cancer (CRC) patients are emerging as biomarkers of acquired resistance to Epidermal Growth Factor Receptor therapy. Unfortunately, reliable assays granting fast, real-time monitoring of treatment response, capable of refining retrospective, tissue-based analysis, are still needed. Recently, several methods for detecting blood RAS mutations have been proposed, generally relying on multi-step and PCR-based, time-consuming and cost-ineffective procedures. By exploiting a liquid biopsy approach, we developed an ultrasensitive nanoparticle-enhanced plasmonic method for detecting ~1 aM RAS single nucleotide variants (SNVs) in the plasma of CRC patients. The assay does not require the extraction of tumor DNA from plasma and detects it in volumes as low as 40 μL of plasma, which is at least an order of magnitude smaller than that required by state of the art liquid biopsy technologies. The most prevalent RAS mutations are detected in DNA from tumor tissue with 100% sensitivity and 83.33% specificity. Spike-in experiments in human plasma further encouraged assay application on clinical specimens. The assay was proven in plasma from CRC patients and healthy donors, and full discrimination between mutated DNA from patients over wild-type DNA from healthy volunteers was obtained thus demonstrating its promising avenue for cancer monitoring based on liquid biopsy. Image 1 • Nanoparticle-enhanced surface plasmon resonance imaging is used to reveal oncogenic DNA with a liquid biopsy approach. • Circulating DNA is detected with a PCR-free approach and simple pre-analytical treatment of the plasma from cancer patients. • Volumes of plasma as low as 40 μL are used, whereas standard technologies require a few hundreds of microliters of plasma. • RAS mutated DNA from tumor tissue is detected with 100% sensitivity and 83.33% specificity. • Full discrimination between mutated DNA from cancer patients over wild-type DNA from healthy volunteers is obtained. [ABSTRACT FROM AUTHOR]

• Antifouling polymer brushes with incorporated aptamer ligands specific to thrombin. • Affinity interaction analysis of three established aptamers on polymer brushes architecture compared to regular thiol SAM. • Superior antifouling properties of functionalized - brushes allowed for direct label-free SPR detection of thrombin. • Implementation of direct detection of thrombin in 10 % whole human blood at clinically relevant concentrations in 15 min. Optical affinity biosensors are pursued for timely monitoring of thrombin in human blood, which is of urgent need in tailored anticoagulation therapies. However, the unspecific deposition of molecules, cells, and aggregates from the blood at their surface (also termed fouling) severely hinders their development and impedes the deploying of this technology to everyday clinical practice. We addressed this challenge by designing surface plasmon resonance (SPR) sensor chip with an antifouling polymer brush architecture and incorporated thrombin aptamer bioreceptors. Poly[(N -(2-hydroxypropyl)-methacrylamide)- co -(carboxybetaine methacrylamide)] brushes were synthesized on gold sensor chip surface via photoinduced single-electron transfer living radical polymerization and postmodified with three thrombin aptamers (HD1 short, HD1 and HD22). The affinity interaction of the aptamer bioreceptors with thrombin (as well as with other molecules present in the blood) was investigated and changes in their performance when incorporated into the polymer brushes were characterized. The combination of brushes and aptamer bioreceptors allowed for the analysis of medically relevant concentrations of thrombin in the 10 % blood by direct SPR detection format. This is the first time that the optical affinity biosensor is demonstrated for label-free analysis of biomarkers in a minimally processed human blood without a need for pre-separation steps. We believe that this system constitutes a basis for the future affinity biosensor applications that are suitable for the clinical settings and can be readily adapted to detect a range of important biological markers. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3′UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3′UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression by targeting mRNAs in a sequence specific manner, thereby determining their degradation or inhibiting translation. They are involved in processes such as proliferation, differentiation and apoptosis by fine-tuning the expression of genes underlying such events. The expression of specific miRNAs is involved in hematopoietic differentiation and their deregulation contributes to the development of hematopoietic malignancies such as acute myeloid leukemia (AML). miR-130a is over-expressed in AML. Here we show that miR-130a is physiologically expressed in myeloblasts and down-regulated during monocyte differentiation. Gain- and loss-of-function experiments performed on CD34+ human hematopoietic stem cells confirmed that expression of miR-130a inhibits monocyte differentiation by interfering with the expression of key transcription factors HOXA10, IRF8, KLF4, MAFB and PU-1. The data obtained in this study highlight that the correct modulation of miR-130a is necessary for normal differentiation to occur and confirming that deregulation of this miRNA might underlie the differentiation block occurring in AML. [ABSTRACT FROM AUTHOR]

MiRNAs are single stranded RNAs of 18-22 nucleotides. They are promising diagnostic and prognostic markers for several pathologies including tumors, neurodegenerative, cardiovascular and autoimmune diseases. In the present work the development and characterization of anti-miRNA radiolabeled probes based on peptide nucleic acids (PNAs) for potential non-invasive molecular imaging in vivo of giant cell arteritis are described. MiR-146a and miR-146b-5p were selected as targets because they have been found up-regulated in this disease. Anti-miR and scramble PNAs were synthesized and linked to carboxyfluorescein or DOTA. DOTA-anti-miR PNAs were then labelled with copper-64 (64Cu) to function as non-invasive molecular imaging tools. The affinity of the probes for the targets was assessed in vitro by circular dichroism and melting temperature. Differential uptake of fluorescein and 64Cu labeled anti-miRNA probes was tested on BCPAP and A549 cell lines, expressing different levels of miR-146a and -146b-5p. The experiments showed that the anti-miR-146a PNAs were more effective than the anti-miR-146b-5p PNAs. Anti-miR-146a PNAs could bind both miR-146a and miR-146b-5p. The uptake of fluorescein and 64Cu labeled anti-miR-146a PNAs was higher than that of the negative control scramble PNAs in miRNA expressing cells in vitro. 64Cu-anti-miR-146a PNAs might be further investigated for non-invasive PET imaging of miR-146 overexpressing diseases. [ABSTRACT FROM AUTHOR]

A new amperometric sandwich-format genosensor has been implemented on single-walled carbon nanotubes screen printed electrodes (SWCNT-SPEs) and compared in terms of performance with analogous genoassays developed using the same methodology on non-nanostructured glassy carbon platforms (GC-SPE). The working principle of the genosensors is based on the covalent immobilization of Peptide Nucleic Acid (PNA) capture probes (CP) on the electrode surface, carried out through the carboxylic functions present on SWCNT-SPEs (carboxylated SWCNT) or electrochemically induced on GC-SPEs. The sequence of the CP was complementary to a 20-mer portion of the target DNA; a second biotin-tagged PNA signalling probe (SP), with sequence complementary to a different contiguous portion of the target DNA, was used to obtain a sandwich hybrid with an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). Comparison of the responses obtained from the SWCNT-SPEs with those produced from the non-nanostructured substrates evidenced the remarkable enhancement effect given by the nanostructured electrode platforms, achieved both in terms of loading capability of PNA probes and amplification of the electron transfer phenomena exploited for the signal transduction, giving rise to more than four-fold higher sensitivity when using SWCNT-SPEs. The nanostructured substrate allowed to reach limit of detection (LOD) of 71 pM and limit of quantitation (LOQ) of 256 pM, while the corresponding values obtained with GC-SPEs were 430 pM and 1.43 nM, respectively. [ABSTRACT FROM AUTHOR]