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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]

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]

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: 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]

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]

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]

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]

Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.

The development of smart nanoparticles (NPs) that encode responsive features in the structural framework promises to extend the applications of NP-based drugs, vaccines, and diagnostic tools. New nanocarriers would ideally consist of a minimal number of biocompatible components and exhibit multiresponsive behavior to specific biomolecules, but progress is limited by the difficulty of synthesizing suitable building blocks. Through a nature-inspired approach that combines the programmability of nucleic acid interactions and sol-gel chemistry, we report the incorporation of synthetic nucleic acids and analogs, as constitutive components, into organosilica NPs. We prepared different nanomaterials containing single-stranded nucleic acids that are covalently embedded in the silica network. Through the incorporation of functional nucleic acids into the organosilica framework, the particles respond to various biological, physical, and chemical inputs, resulting in detectable physicochemical changes. The one-step bottom-up approach used to prepare organosilica NPs provides multifunctional systems that combine the tunability of oligonucleotides with the stiffness, low cost, and biocompatibility of silica for different applications ranging from drug delivery to sensing.

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.

Peptide nucleic acids (PNAs) are a class of artificial oligonucleotide mimics that have garnered much attention as precision biotherapeutics for their efficient hybridization properties and their exceptional biological and chemical stability. However, the poor cellular uptake of PNA is a limiting factor to its more extensive use in biomedicine; encapsulation in nanoparticle carriers has therefore emerged as a strategy for internalization and delivery of PNA in cells. In this study, we demonstrate that PNA can be readily loaded into porous silicon nanoparticles (pSiNPs) following a simple salt-based trapping procedure thus far employed only for negatively charged synthetic oligonucleotides. We show that the ease and versatility of PNA chemistry also allows for producing PNAs with different net charge, from positive to negative, and that the use of differently charged PNAs enables optimization of loading into pSiNPs. Differently charged PNA payloads determine different release kinetics and allow modulation of the temporal profile of the delivery process. In vitro silencing of a set of specific microRNAs using a pSiNP-PNA delivery platform demonstrates the potential for biomedical applications.

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant ( K D ) value of 43 ± 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available.

Standard protocols for the analysis of circulating tumor DNA (ctDNA) include the isolation of DNA from the patient's plasma and its amplification and analysis in buffered solutions. The application of such protocols is hampered by several factors, including the complexity and time-constrained preanalytical procedures, risks for sample contamination, extended analysis time, and assay costs. A recently introduced nanoparticle-enhanced surface plasmon resonance imaging-based assay has been shown to simplify procedures for the direct detection of tumor DNA in the patient's plasma, greatly simplifying the cumbersome preanalytical phase. To further simplify the protocol, a new dual-functional low-fouling poly-l-lysine (PLL)-based surface layer has been introduced that is described herein. The new PLL-based layer includes a densely immobilized CEEEEE oligopeptide to create a charge-balanced system preventing the nonspecific adsorption of plasma components on the sensor surface. The layer also comprises sparsely attached peptide nucleic acid probes complementary to the sequence of circulating DNA, e.g., the analyte that has to be captured in the plasma from cancer patients. We thoroughly investigated the contribution of each component of the dual-functional polymer to the antifouling properties of the surface layer. The low-fouling property of the new surface layer allowed us to detect wild-type and KRAS p.G12D-mutated DNA in human plasma at the attomolar level (∼2.5 aM) and KRAS p.G13D-mutated tumor DNA in liquid biopsy from a cancer patient with almost no preanalytical treatment of the patient's plasma, no need to isolate DNA from plasma, and without PCR amplification of the target sequence.

A novel synthesis of C(2)-modified peptide nucleic acids (PNAs) is proposed, using a submonomeric strategy with minimally protected building blocks, which allowed a reduction in the required synthetic steps. N(3)-unprotected, d-Lys- and d-Arg-based backbones were used to obtain positively charged PNAs with high optical purity, as inferred from chiral GC measurements. "Chiral-box" PNAs targeting the G12D point mutation of the KRAS gene were produced using this method, showing improved sequence selectivity for the mutated- vs wild-type DNA strand with respect to unmodified PNAs.

The importance of peptide nucleic acids (PNAs) for alteration of gene expression is nowadays firmly established. PNAs are characterized by a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units and have been found to be excellent candidates for antisense and antigene therapies. Recently, PNAs have been demonstrated to alter the action of microRNAs and thus can be considered very important tools for miRNA therapeutics. In fact, the pharmacological modulation of microRNA activity appears to be a very interesting approach in the development of new types of drugs. Among the limits of PNAs in applied molecular biology, the delivery to target cells and tissues is of key importance. The aim of this chapter is to describe methods for the efficient delivery of unmodified PNAs designed to target microRNAs involved in cancer, using as model system miR-221-3p and human glioma cells as in vitro experimental cellular system. The methods employed to deliver PNAs targeting miR-221-3p here presented are based on a macrocyclic multivalent tetraargininocalix[4]arene used as non-covalent vector for anti-miR-221-3p PNAs. High delivery efficiency, low cytotoxicity, maintenance of the PNA biological activity, and easy preparation makes this vector a candidate for a universal delivery system for this class of nucleic acid analogs.

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.

The available active surface area and the density of probes immobilized on this surface are responsible for achieving high specificity and sensitivity in electrochemical biosensors that detect biologically relevant molecules, including DNA. Here, we report the design of gold-coated, silicon micropillar-structured electrodes functionalized with modified poly-l-lysine (PLL) as an adhesion layer to concomitantly assess the increase in sensitivity with the increase of the electrochemical area and control over the probe density. By systematically reducing the center-to-center distance between the pillars (pitch), denser micropillar arrays were formed at the electrode, resulting in a larger sensing area. Azido-modified peptide nucleic acid (PNA) probes were click-reacted onto the electrode interface, exploiting PLL with appended oligo(ethylene glycol) (OEG) and dibenzocyclooctyne (DBCO) moieties (PLL-OEG-DBCO) for antifouling and probe binding properties, respectively. The selective electrochemical sandwich assay formation, composed of consecutive hybridization steps of the target complementary DNA (cDNA) and reporter DNA modified with the electroactive ferrocene functionality (rDNA-Fc), was monitored by quartz crystal microbalance. The DNA detection performance of micropillared electrodes with different pitches was evaluated by quantifying the cyclic voltammetric response of the surface-confined rDNA-Fc. By decrease of the pitch of the pillar array, the area of the electrode was enhanced by up to a factor 10.6. A comparison of the electrochemical data with the geometrical area of the pillared electrodes confirmed the validity of the increased sensitivity of the DNA detection by the design of the micropillar array.

The immobilization of biomolecules onto polymeric surfaces employed in the fabrication of biomedical and biosensing devices is generally a challenging issue, as the absence of functional groups in such materials does not allow the use of common surface chemistries. Here we report the use of modified poly-l-lysine (PLL) as an effective method for the selective modification of polymeric materials with biomolecules. Cyclic olefin polymer (COP), Ormostamp, and polydimethylsiloxane (PDMS) surfaces were patterned with modified PLLs displaying either biotin or maleimide functional groups. Different patterning techniques were found to provide faithful microscale pattern formation, including micromolding in capillaries (MIMIC) and a hydrogel-based stamping device with micropores. The surface modification and pattern stability were tested with fluorescence microscopy, contact angle and X-ray photoelectron spectroscopy (XPS), showing an effective functionalization of substrates stable for over 20 days. By exploiting the strong biotin-streptavidin interaction or the thiol-maleimide coupling, DNA and PNA probes were displayed successfully on the surface of the materials, and these probes maintained the capability to specifically recognize complementary DNA sequences from solution. The printing of three different PNA-thiol probe molecules in a microarray fashion allowed selective DNA detection from a mixture of DNA analytes, demonstrating that the modified PLL methodology can potentially be used for multiplexed detection of DNA sequences., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

The present study investigated the effects of the combined treatment of two peptide nucleic acids (PNAs), directed against microRNAs involved in caspase‑3 mRNA regulation (miR‑155‑5p and miR‑221‑3p) in the temozolomide (TMZ)‑resistant T98G glioma cell line. These PNAs were conjugated with an octaarginine tail in order to obtain an efficient delivery to treated cells. The effects of singularly administered PNAs or a combined treatment with both PNAs were examined on apoptosis, with the aim to determine whether reversion of the drug‑resistance phenotype was obtained. Specificity of the PNA‑mediated effects was analyzed by reverse transcription‑quantitative polymerase‑chain reaction, which demonstrated that the effects of R8‑PNA‑a155 and R8-PNA-a221 anti‑miR PNAs were specific. Furthermore, the results obtained confirmed that both PNAs induced apoptosis when used on the temozolomide‑resistant T98G glioma cell line. Notably, co‑administration of both anti‑miR‑155 and anti‑miR‑221 PNAs was associated with an increased proapoptotic activity. In addition, TMZ further increased the induction of apoptosis in T98G cells co‑treated with anti‑miR‑155 and anti‑miR‑221 PNAs.

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 ( 64 Cu) 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 64 Cu 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 64 Cu labeled anti-miR-146a PNAs was higher than that of the negative control scramble PNAs in miRNA expressing cells in vitro. 64 Cu-anti-miR-146a PNAs might be further investigated for non-invasive PET imaging of miR-146 overexpressing diseases.

Biosensors and materials for biomedical applications generally require chemical functionalization to bestow their surfaces with desired properties, such as specific molecular recognition and antifouling properties. The use of modified poly(l-lysine) (PLL) polymers with appended oligo(ethylene glycol) (OEG) and thiol-reactive maleimide (Mal) moieties (PLL-OEG-Mal) offers control over the presentation of functional groups. These reactive groups can readily be conjugated to, for example, probes for DNA detection. Here we demonstrate the reliable conjugation of thiol-functionalized peptide nucleic acid (PNA) probes onto predeposited layers of PLL-OEG-Mal and the control over their surface density in the preceding synthetic step of the PLL modification with Mal groups. By monitoring the quartz crystal microbalance (QCM) frequency shifts of the binding of complementary DNA versus the density of Mal moieties grafted to the PLL, a linear relationship between probe density and PLL grafting density was found. Cyclic voltammetry experiments using Methylene Blue-functionalized DNA were performed to establish the absolute probe density values at the biosensor surfaces. These data provided a density of 1.2 × 10 12 probes per cm 2 per % of grafted Mal, thus confirming the validity of the density control in the synthetic PLL modification step without the need of further surface characterization.

Silicon nanowire chips can function as sensors for cancer DNA detection, whereby selective functionalization of the Si sensing areas over the surrounding silicon oxide would prevent loss of analyte and thus increase the sensitivity. The thermal hydrosilylation of unsaturated carbon-carbon bonds onto H-terminated Si has been studied here to selectively functionalize the Si nanowires with a monolayer of 1,8-nonadiyne. The silicon oxide areas, however, appeared to be functionalized as well. The selectivity toward the Si-H regions was increased by introducing an extra HF treatment after the 1,8-nonadiyne monolayer formation. This step (partly) removed the monolayer from the silicon oxide regions, whereas the Si-C bonds at the Si areas remained intact. The alkyne headgroups of immobilized 1,8-nonadiyne were functionalized with PNA probes by coupling azido-PNA and thiol-PNA by click chemistry and thiol-yne chemistry, respectively. Although both functionalization routes were successful, hybridization could only be detected on the samples with thiol-PNA. No fluorescence was observed when introducing dye-labeled noncomplementary DNA, which indicates specific DNA hybridization. These results open up the possibilities for creating Si nanowire-based DNA sensors with improved selectivity and sensitivity.

Peptide Nucleic Acids (PNAs) are oligonucleotide mimics that can be used as drugs as they can interact with DNA and RNA targets in organisms. Loading PNAs into inorganic nanocarriers can improve their cellular uptake and co-delivering them together with drugs can improve the therapy efficacy by synergic effects. Furthermore, the functionalization of the carriers with labels allows theranostics, and the possibility to monitor the efficacy of the therapy in real time. The present protocol describes the synthesis of Zeolites-L nanocrystals and mesoporous silica nanoparticles and their loading with cationic PNAs and other smaller molecular weight payloads towards theranostics applications.

Peptide Nucleic Acids (PNAs) are oligonucleotide mimics that can be used to block the biological action of microRNA, thus affecting gene expression post-transcriptionally. PNAs are obtained with solid-phase peptide synthesis, and can be easily conjugated to other peptides. Conjugation with R8-Peptide or modification of the PNA backbone (at C5 or C2 carbon) with arginine side chains allows efficient cellular uptake. The present protocol describes the synthesis of cationic PNAs that can be used alone as drugs or for efficient co-delivery in suitable inorganic nanocarriers.

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., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Mesoporous silica nanoparticles (MSNPs), 100 nm in size, incorporating a Cy5 fluorophore within the silica framework, are synthesized and loaded with the anti-cancer drug temozolomide (TMZ), used in the treatment of gliomas. The surface of the particles is then decorated, using electrostatic interactions, with a polyarginine-peptide nucleic acid (R8-PNA) conjugate targeting the miR221 microRNA. The multi-functional nanosystem thus obtained is rapidly internalized into glioma C6 or T98G cells. The anti-miR activity of the PNA is retained, as confirmed by reverse transcription polymerase chain reaction (RT-PCR) measurements and induction of apoptosis is observed in temozolomide-resistant cell lines. The TMZ-loaded MSNPs show an enhanced pro-apoptotic effect, and the combined effect of TMZ and R8-PNA in the MSNPs shows the most effective induction of apoptosis (70.9% of apoptotic cells) thus far achieved in the temozolomide-resistant T98G cell line., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The design and synthesis of smart nanomaterials can provide interesting potential applications for biomedical purposes from bioimaging to drug delivery. Manufacturing multifunctional systems in a way to carry bioactive molecules, like peptide nucleic acids able to recognize specific targets in living cells, represents an achievement towards the development of highly selective tools for both diagnosis and therapeutics. This work describes a very first example of the use of zeolite nanocrystals as multifunctional nanocarriers to deliver simultaneously PNA and organic molecules into living cells. Zeolite-L nanocrystals are functionalized by covalently attaching the PNA probes onto the surface, while the channel system is filled with fluorescent guest molecules. The cellular uptake of the PNA/Zeolite-L hybrid material is then significantly increased by coating the whole system with a thin layer of biodegradable poly-L-lysine. The delivery of DAPI as a model drug molecule, inserted into the zeolite pores, is also demonstrated to occur in the cells, proving the multifunctional ability of the system. Using this zeolite nanosystem carrying PNA probes designed to target specific RNA sequences of interest in living cells could open new possibilities for theranostic and gene therapy applications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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

Peptide nucleic acids (PNAs) are synthetic oligonucleotide analogues based on a pseudopeptide backbone that bind complementary DNA or RNA with high affinity and specificity. In this chapter, three PNA-based genotyping assays are described: PCR clamping, fluorescence-based recognition, and microarray platform. The first two methods are performed in solution, while the microarray method uses a solid surface.

Chiral open-chain PNAs have been shown to have improved properties in terms of control of helical handedness, DNA affinity, sequence selectivity, and cellular uptake. They can be synthesized either using preformed chiral monomers or by means of a submonomeric strategy. The former is preferred when only a stereogenic center is present at C-5, whereas for PNA-bearing substituents at C-2, the submonomeric approach is preferred, since racemization, generally occurring during the solid-phase synthesis, can be minimized by this procedure. Here we describe the protocols for the synthesis of PNA oligomers containing C-2- or C-5- (or both) modified monomers and a GC method for checking the optical purity of C-2-modified PNAs.

The involvement of microRNAs in human pathologies is a firmly established fact. Accordingly, the pharmacological modulation of their activity appears to be a very appealing issue in the development of new types of drugs (miRNA therapeutics). One of the most interesting issues is the possible development of miRNA therapeutics for development of anti-cancer molecules. In this respect appealing molecules are based on peptide nucleic acids (PNAs), displaying a pseudo-peptide backbone composed of N-(2-aminoethyl)glycine units and found to be excellent candidates for antisense and antigene therapies. The major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells. The aim of this chapter is to describe methods for determining the activity of PNAs designed to target oncomiRNAs, using as model system miR-221 and its target p27(Kip1) mRNA. The effects of PNAs targeting miR-221 are here presented discussing data obtained using as model system the human breast cancer cell line MDA-MB-231, in which miR-221 is up-regulated and p27(Kip1) down-regulated.

Peptide nucleic acids (PNAs) are DNA/RNA mimics extensively used for pharmacological regulation of gene expression in a variety of cellular and molecular systems, and they have been described as excellent candidates for antisense and antigene therapies. At present, very few data are available on the use of PNAs as molecules targeting miRNAs. miRNAs are a family of small nc RNAs that regulate gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation to the control of highly regulated biological functions, such as differentiation, cell cycle and apoptosis. The aim of this article is to present the state-of-the-art concerning the possible use of PNAs to target miRNAs and modify their biological metabolism within the cells. The results present in the literature allow to propose PNA-based molecules as very promising reagents to modulate the biological activity of miRNAs. In consideration of the involvement of miRNAs in human pathologies, PNA-mediated targeting of miRNAs has been proposed as a potential novel therapeutic approach.

PNAs (peptide nucleic acids) have been immobilized onto surfaces in a fast, accurate way by employing reactive microcontact printing. Surfaces have been first modified with aldehyde groups to react with the amino end of the synthesized PNAs. When patterning fluorescein-labeled PNAs by reactive microcontact printing using oxygen-oxidized polydimethylsiloxane stamps, homogeneous arrays were fabricated and characterized using optical methods. PNA-patterned surfaces were hybridized with complementary and mismatched dye-labeled oligonucleotides to test their ability to recognize DNA sequences. The stability and selectivity of the PNA-DNA duplexes on surfaces have been verified by fluorescence microscopy, and the melting curves have been recorded. Finally, the technique has been applied to the fabrication of chips by spotting a PNA microarray onto a flat PDMS stamp and reproducing the same features onto many slides. The chips were finally applied to single nucleotide polymorphism detection on oligonucleotides.

Helical handedness and the twist and tilt parameters of the base pairs in duplex DNA can be affected by base sequence variation and change in environmental conditions as occurs in the transformation between right-handed B-DNA and left-handed Z-DNA. For duplexes of DNA with oligonucleotide analogs such as peptide nucleic acids (PNAs), less is known about the effects on structure such as the base pair twist and tilt parameters and handedness. However, in PNA:PNA duplexes, the absence of chiral information determining helical handedness allows the relationship between preferred helical handedness and structural design to be manipulated and, therefore, better understood. In this chapter, we report a protocol for switching between B- and Z-DNA:DNA duplexes, and the experimental procedures for obtaining right- or left-handed PNA:PNA duplexes.

Enantiomeric analysis is one of the crucial points for the sensor technology, due to the increasing importance that enantiomerically pure compounds and drugs have in pharmaceutic and agrochemical applications. Enantiomeric luminescent sensors give different responses by interaction or reaction with chiral molecules, allowing one to assess their optical purity by spectroscopic measurements. Moreover, chemosensors have been developed to perform enantiomeric analysis of both luminescent and non-luminescent organic compounds. In the present chapter we focus on the recent advances in the sensing of chiral molecules by luminescent sensory systems, with the aim of outlining different mechanisms: fluorescence quenching by metal complexes, photoinduced electron transfer (PET) quenching, fluorescence enhancement by PET inhibition, analyte induced sensor conformational changes, modulation of excimer and exciplex formation, and aggregation induced emission enhancement (AIEE). Recent advances in the use of more elaborate techniques such as anisotropy measurements, gated detection, circularly polarized luminescence (CPL) and perspectives in the field are also discussed. Emphasis is given to the methods which have provided high enantioselectivity and which are amenable to fast screening procedures.

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 41 zM sensitivity in targeting genomic DNA even in the presence of a large excess of non-complementary DNA., (2010 Elsevier B.V. All rights reserved.)

The synthesis of C5 linked uracil dimers was carried out according to a model developed in order to bind adenine in DNA. N1-Alkylated uracil derivatives were synthesized from isoorotic acid (uracil-5-carboxylic acid) or thymine. The carboxylic acid derivatives were condensed with diamines in order to produce dimeric compounds or with monoamines in order to obtain reference monomeric compounds. Some of the derivatives, in particular the uracil dimers, showed antiproliferative and erythroid differentiation induction properties towards human chronic myelogenous leukemia K562 cells, thus indicating that these compounds could represent a new class of drugs useful for the development of antitumor therapy based on the ability to induce terminal differentiation.

In order to recognize the presence of the R553X point mutation of the cystic fibrosis (CF) gene in the human genome, a peptide nucleic acid (PNA) complementary to the mutated gene tract and bearing three adjacent chiral monomers based on D-lysine (chiral box) was synthesized and used as a probe in CE. Binding specificity was preliminarily studied with complementary and mismatched oligonucleotides by UV spectroscopy, electrospray MS, and electrophoresis, indicating a very high sequence selectivity. The chiral PNA probe was then hybridized to cyanine-5-labeled DNA samples (186 bp), obtained by PCR amplification, respectively, from: (a) normal homozygous subjects (wtDNA), (b) CF-affected homozygous subjects (mutDNA), (c) heterozygous subjects (healthy carriers) and denatured at low ionic strength. The PNA-DNA mixture was directly analyzed by CE with LIF detection: a new signal corresponding to the PNA-mutDNA duplex was observed, in the case of CF-affected homozygous subjects, whereas for the sample containing the mismatched sequence (normal homozygous wtDNA) only the signal corresponding to ssDNA (ss, single strand) was detected. In the case of heterozygous DNA, both PNA-mutDNA duplex and ssDNA were detected. With this simple assay, it was possible to discriminate in an easy way among the three cases (mutated homozygous, normal homozygous, and heterozygous subjects) with a total specificity, thus allowing a decisive advance for the diagnosis of CF.

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance., (Copyright 2003 John Wiley & Sons, Ltd.)