Roberto Corradini

Occurrence of deoxynivalenol and its 3-β-D-glucoside in wheat and maize

Deoxynivalenol-3-β-D-glucoside (D3G), a phase II plant metabolite of the mycotoxin deoxynivalenol (DON), occurs in naturally Fusarium-contaminated cereals. In order to investigate the frequency of occurrence as well as the relative and absolute concentrations of D3G in naturally infected cereals, 23 wheat samples originating from fields in Austria, Germany and Slovakia as well as 54 maize samples from Austrian fields were analysed for DON and D3G by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both analytes were detected in all the 77 field samples. DON was found at levels from 42 to 4130 ng g−1 (977 ± 1000 ng g−1 on average). The D3G concentrations in all cereal samples were in the range 10–1070 ng g−1 (216 ± 253 ng g−1 on average), corresponding to about 5–46 mol% of their DON concentrations (15 ± 8 mol% on average).

Insights into peptide nucleic acid (PNA) structural features: The crystal structure of a D-lysine-based chiral PNA-DNA duplex

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton.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-Å resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. Thed-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.

DNA Binding of AD-Lysine-Based Chiral PNA: Direction Control and Mismatch Recognition

Peptide nucleic acids (PNAs) are oligonucleotide analogues with a skeleton made up of N-(2-aminoethyl)glycine units; they bind to complementary DNA and RNA with high stability and specificity. In order to improve the binding specificity, solubility and uptake into cells, many modifications have been introduced, some concerning the introduction of stereogenic centres. With the aim of achieving a selective antiparallel binding with DNA, we report in this paper the synthesis and binding abilities of a chiral PNA decamer (H-GTAGATCACT-NH2) bearing three D-Lys-based monomers (a “chiral box”) in the middle of the strand. Indeed, the antiparallel PNA-DNA duplex showed a melting point of 43 °C (determined both by CD and UV spectroscopy), whereas the parallel PNA-DNA duplex failed to form, as shown by the absence of temperature dependence in the UV and CD spectra. Moreover, hybridization experiments carried out with antiparallel DNA strands bearing single mismatches showed that this PNA was excellent in discriminating between mismatched and matched targets. These results indicate that a high chiral constraint in the middle of a PNA sequence strongly affects the direction selectivity, i.e. the antiparallel/parallel preference in the DNA complexation. In particular, a “D-chiral box” favours a highly specific antiparallel DNA binding, thus allowing possible diagnostic applications for the screening of single-point mutations.

Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development.

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.

Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis

We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH2 terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G1-phase accumulation (39–53%) in IMR-32 and apoptosis (56% annexin V–positive cells at 24 hours in IMR-32 and 22% annexin V–positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA–based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression.

Ultrasensitive detection of non-amplified genomic DNA by nanoparticle-enhanced surface plasmon resonance imaging.

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.

Ultrasensitive Detection of DNA by PNA and Nanoparticle-Enhanced Surface Plasmon Resonance Imaging

Materials and reagents: Reagents were obtained from commercial suppliers and used without further purification. Wild-type streptavidin (WT-SA) was purchased from Invitrogen (Italy). Nitrocellulose membrane filters was purchased from Whatman (U.K.). Trisodium citrate dihydrate, tetrachloroauric(III) acid (HAuCl4·3H2O), triethanolamine (TEA), ethanol, dimethyl sulfoxide (DMSO), hexane, sodium hydroxide solutions (10 M in water), dithiobis(N)succinimidylpropionate (Lomants Reagent or DTSP) were purchased from Sigma-Aldrich (Italy). Methoxy-polyethyleneglycol amine (mPEG-NH2, MW = 5000) was purchased from Nektar Therapeutics (USA). Phosphate-buffered saline (PBS) solutions at pH 7.4, (NaCl 137 mM, 2.7 mM KCl, phosphate-buffered 10 mM) was obtained from Amresco (Italy). SPRI gold chips were purchased from GWC Technologies (USA). Ultra-pure water (Milli-Q Element, Millipore) was used for all the experiments. Microfluidic devices fabrication: Two different microfluidic devices with parallel and Y-shaped microchannels respectively were used for the study. Both of them were fabricated in poly(dimethyl siloxane) (PDMS) polymer through the well established replica molding technique elsewhere described. [1] Briefly, PDMS channels were creat

Enantioselective Sensing by Luminescence

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.

Toward A Highly Specific DNA Biosensor: PNA-Modified Suspended-Core Photonic Crystal Fibers

The feasibility of a biosensor for DNA detection based on suspended-core photonic crystal fibers is investigated. The possibility of functionalization of the hole surface, which allows DNA strand binding, is demonstrated, along with the selective detection of DNA through hybridization of immobilized peptide nucleic acid probes with their full-complementary and mismatched DNA segments.

Dansylated Polyamines as Fluorescent Sensors for Metal Ions: Photophysical Properties and Stability of Copper(II) Complexes in Solution

Protonation and the CuII complexation constants of the dansylated polyamines N-dansylethylenediamine (1), N-dansyldiethylenetriamine (2), N-dansyltriethylenetetramine (3), N′-[2-(dansylamino)ethyl]diethylenetriamine (4), and tris(2-dansylaminoethyl)amine (5) were determined by glass-electrode potentiometry in MeOH/H2O 9 : 1 (v/v) solution. For ligands 3 and 4, the determinations were also performed in aqueous solution. The complexes formed by these ligands in neutral form correspond to those observed for the analogous unsubstituted monoprotonated amines, whereas, when the ligands are deprotonated at the sulfonamide moiety, the species parallel those of the corresponding amines. The molecular structures of the complexes were deduced from the VIS absorption spectra. The crystal structure of the [CuL2H−2] complex 6 of ligand 1 (L) was determined by X-ray diffraction. The study of the photophysical properties of the ligands 3 – 5 showed that they are good fluorescent sensors for copper(II), which induced fluorescence quenching. Time-resolved fluorescence measurements allowed us to clarify the sensing mechanism. The pH dependence of the quenching effect demonstrated that it occurs for all Cu2+ complexes, even for species in which the sulfonamide moiety is not deprotonated. Sensing of Cu2+ was compared with that of other metal ions (Co2+, Ni2+, Zn2+, Cd2+, Hg2+), and selectivity was studied as a function of pH. Ligands 3 and 4 were found to be selective chemosensors for Cu2+ in weakly acidic solution (pH ca. 4 – 5).