Biomedical subjects

Roberto Corradini

Publications and source records attributed to Roberto Corradini.

18 recordsLinked to original sources

PubMed2006

Highly efficient strand invasion by peptide nucleic acid bearing optically pure lysine residues in its backbone.

Chiral PNA monomers (PNA = peptide nucleic acid), in which nucleobases are attached to N-(aminoethyl)-D-lysine, were introduced to PNAs bearing pseudo-complementary nucleobases (2,6-diaminopurine and 2-thiouracil). When these highly cationic PNAs targeted double-stranded DNA, they invaded there much more efficiently than conventional pseudo-complementary PNAs composed of achiral PNA monomers. Although introduction of N-(aminoethyl)-D-lysine backbone was effective for promotion of strand invasion, L-isomer never promote it. Simple incorporation of lysine groups to the termini of PNA was also ineffective, indicating that introduction of positive charges into PNA backbone is important. Even highly G-C rich sequence, which conventional pseudo-complementary PNAs never invade, was successfully targeted based on this strategy.

2-Aminopurine↗

PubMed2005

Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food.

Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.

DNA, Plant↗

PubMed2005

Lysine-based peptide nucleic acids (PNAs) with strong chiral constraint: control of helix handedness and DNA binding by chirality.

Two enantiomeric chiral PNAs bearing three adjacent D- or L-lysine-based residues in the middle of the strand ("chiral box" PNAs, sequence H-GTAGA(Lys)T(Lys)C(Lys)ACT-NH2) have been used as models in order to comprehensively study the effects of the stereogenic centers on PNA conformation and on PNA binding properties to complementary PNA and DNA strands. The binding properties of the two enantiomeric PNAs and of their homologous achiral PNA have been extensively studied by UV and CD spectroscopy and by mass spectrometry, both in the antiparallel and in the parallel mode with complementary PNA and DNA strands. In the antiparallel PNA:PNA duplexes, L-Lys PNA were found to form left-handed, and D-Lys PNA right handed helices, while in parallel duplexes, the reversed helicities were observed. Correspondingly, the preferred mode of binding and the best mismatch recognition of the D-Lys containing PNA with (right handed) DNA was found to be in the antiparallel orientation, while that of L-Lys PNA was found to be in the parallel mode. A rationale which correlates the preferred handedness of the PNA-PNA duplexes to the directionality of the binding to complementary DNA duplexes has been devised according to structural data and considering the "retro-inverso" concept widely used for peptides.

Base Pair Mismatch↗

PubMed2005

Unconventional method based on circular dichroism to detect peanut DNA in food by means of a PNA probe and a cyanine dye.

In this paper we report an innovative and unconventional method based on circular dichroism for the identification of peanut DNA in food, which can be detected after PCR amplification at the nanomolar level by using an achiral PNA probe complementary to a tract of the peanut Ara h 2 gene and an achiral 3,3'-diethylthiadicarbocyanine dye [DiSC(2)(5)]. Peanuts are one of the most common causes of severe allergic reactions to foods and are particularly dangerous when they are "hidden" (undeclared) in food. For better protection of consumers, detection methods are required to specifically detect the presence of hidden allergens in a wide variety of food items. Alternative to the detection of the proteins is the determination of species-specific DNA, which is more resistant to technological treatments. PNAs are very specific probes able to recognize DNA sequences with high affinity and evidence for the binding can be obtained by using the DiSC(2)(5) dye, which aggregates onto the PNA-DNA duplex giving rise to a characteristic visibile band at 540 nm. Because the PNA-DNA duplex is in a right-handed helical conformation, the aggregation of the dye to the duplex gives also rise to a strong CD signal in the 500-600 nm region with a strong exciton coupling due to the formation of multimeric species, since the handedness of the helix is transferred to the dye aggregate. The dye does not interact with the free single-stranded DNA and although aggregating on the achiral PNA, this interaction is obviously not detectable by circular dichroism. Thus, only the formation of the PNA-DNA duplex, which takes place only upon specific Watson-Crick hydrogen binding between the PNA and the DNA bases, is detected, ensuring a very high specificity and sensitivity. The method has been optimized in a model system by using a synthetic oligonucleotide complementary to the PNA probe, showing that the intensity of the signal is linearly related to the amount of the DNA. The optimized method has been applied to the identification and quantitation of DNA extracted and amplified by PCR from peanuts and from peanut-containing foods, allowing for a very sensitive detection at a very low level (few pmol).

Arachis↗

PubMed2005

Detection of the R553X DNA single point mutation related to cystic fibrosis by a "chiral box" D-lysine-peptide nucleic acid probe by capillary electrophoresis.

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.

Cystic Fibrosis↗

PubMed2005

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 NH(2) 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 G(1)-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.

Apoptosis↗

PubMed2004

Calcein-AM is a detector of intracellular oxidative activity.

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 information 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.

Cell-Free System↗

PubMed2004

Detection of genetically modified soybean using peptide nucleic acids (PNAs) and microarray technology.

Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.

DNA, Plant↗

PubMed2004

Enantioselective fluorescence sensing of amino acids by modified cyclodextrins: role of the cavity and sensing mechanism.

Two selectors based on modified cyclodextrins containing a metal binding site and a dansyl fluorophore-6-deoxy-6-N-(N(alpha)-[(5-dimethylamino-1-naphthalenesulfonyl)aminoethyl]phenylalanylamino-beta-cyclodextrin-containing D-Phe (3) and L-Phe (4) moieties were synthesized. The conformations of the two selectors were studied by circular dichroism, two-dimensional NMR spectroscopy and time-resolved fluorescence spectroscopy. Cyclodextrin 4 was found to have a predominant conformation in which the dansyl group is self-included in the cyclodextrin cavity, while 3 showed a larger proportion of the conformation with the dansyl group outside the cavity. As a consequence, the two cyclodextrins were found to bind copper(II) with different affinities, as revealed by fluorescence quenching in competitive binding measurements. Addition of D- or L-amino acids induced increases in fluorescence intensity, which were dependent on the amino acid used and in some cases on its absolute configuration. The cyclodextrin 4 was found to be more enantioselective than 3, suggesting that the self-inclusion in the cyclodextrin cavity strongly increases the chiral discrimination ability of the copper(II) complex. Accordingly, a linear fluorescent ligand N(alpha)-[(5-dimethylamino-1-naphthalenesulfonyl)aminoethyl]-N(1)-propyl-phenylalaninamide, which has the same binding site and absolute configuration as 4, showed very low chiral discrimination ability. The enantioselectivity in fluorescence response was found to be due to the formation of diastereomeric ternary complexes, which were detected by ESI-MS and by circular dichroism. Time-resolved fluorescence studies showed that the fluorescence of the dansyl group was completely quenched in the ternary complexes formed, and that the residual fluorescence was due to uncomplexed ligand.

Amino Acids↗

PubMed2004

Functional dissection of RNA polymerase III termination using a peptide nucleic acid as a transcriptional roadblock.

We have shown previously that a T(10) peptide nucleic acid (PNA) bound to the transcriptional terminator of a Saccharomyces cerevisiae tDNA(Ile)(TAT) gene arrests elongating yeast RNA polymerase (pol) 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 tDNA(Ile) transcriptional terminator (T(6)) that is not bound by the T(10) 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 bp downstream of the T(6) 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 pol 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 (T(3)GT(2)), 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 (T(2)G(2)T(2) and TG(2)TGT). 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.

Base Sequence↗

PubMed2004

Targeted inhibition of NMYC by peptide nucleic acid in N-myc amplified human neuroblastoma cells: cell-cycle inhibition with induction of neuronal cell differentiation and apoptosis.

We developed an antisense peptide nucleic acid (PNA) targeted against a unique sequence in the terminus of the 5'-UTR of N-myc, designed for selective inhibition of NMYC in neuroblastoma cells. Fluorescent microscopy showed carrier-free delivery of the PNA to two human neuro-blastoma cell lines: GI-LI-N (N-myc-amplified) and GI-CA-N (N-myc-unamplified). Only in the former, PNA treatment determined 70% cell-viability reduction (at 48 h). In N-myc-amplified GI-LI-N cells, the PNA determined NMYC-translation inhibition (Western blotting), accumulation of cells in G1, induction of differentiation and apoptosis. Selectivity of the PNA was demonstrated by altering three point mutations. These findings should encourage development of a PNA-based tumor-specific agent for neuroblastoma (or other neoplasms) with N-myc overexpression.

Apoptosis↗

PubMed2003

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-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.

Base Sequence↗

PubMed2003

Direction control in DNA binding of chiral D-lysine-based peptide nucleic acid (PNA) probed by electrospray mass spectrometry.

The DNA binding abilities of peptide nucleic acids (PNAs), both achiral and bearing three adjacent D-lysine-based monomers in the middle of the strand ("chiral box" PNA), were studied by means of electrospray mass spectrometry (ESI-MS). In contrast with achiral PNA, "Chiral box" PNA was confirmed to exert high direction control (antiparallel vs. parallel DNA target) in DNA binding.

DNA↗

PubMed2003

Enantiomeric separation of chiral peptide nucleic acid monomers by capillary electrophoresis with charged cyclodextrins.

Direct chiral separation of chiral peptide nucleic acid (PNA) monomers has been achieved for the first time by capillary electrophoresis (CE) with charged cyclodextrins as chiral selectors added to the electrophoretic buffer. Selectively modified 6-deoxy-6-N-histamino-beta-cyclodextrin and sulfobutyl ether-beta-CD were successfully used as chiral selectors for the enantiomeric separation of chiral monomers based on different aminoethylamino acids bearing thymine or adenine as nucleobases. Chiral separations were obtained at low selector concentrations (1-3 mM) with good enantioselectivity and resolution factors. Separations were optimized as a function of pH in order to exploit the effect of the electrostatic interactions between the oppositely charged selector and selectand. The method has been applied to the analysis of the enantiomeric excess of chiral monomers used for the solid phase synthesis of chiral PNA oligomers. CE chiral analysis showed that a very high enantiomeric purity was generally achieved in the synthesis of all monomers, except for histidine and aspartic acid based monomers in which ca. 10% of the "wrong" enantiomer was always present.

Cyclodextrins↗

PubMed2003

ESI-mass spectrometry analysis of unsubstituted and disubstituted beta-cyclodextrins: fragmentation mode and identification of the AB, AC, AD regioisomers.

The study of unsubstituted and disubstituted beta-cyclodextrins (beta-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 beta-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-beta-CDs and ditosyl-beta-CD and on the AC and AD regioisomers of dimesitylenesulphonyl-beta-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 beta-CDs.

Cyclodextrins↗

PubMed2002

Crystallization and preliminary X-ray diffraction studies of a D-lysine-based chiral PNA-DNA duplex.

A 10-mer duplex formed between a PNA containing a 'chiral box' of three adjacent D-Lys-based monomers and its complementary DNA strand has been crystallized for the first time. Crystals have been obtained using PEG 8000 as precipitant and cacodylate at pH 6.3 as buffer. The crystals belong to the space group P3(1) or to its enantiomorph P3(2), with unit-cell parameters a = b = 35.00, c = 35.91 A. A complete data set has been collected at the synchrotron source Elettra in Trieste to 1.85 A resolution, using a single frozen crystal.

Crystallization↗

PubMed2002

Role of chirality and optical purity in nucleic acid recognition by PNA and PNA analogs.

Peptide nucleic acids are DNA mimics able to form duplexes with complementary DNA or RNA strands of remarkable affinity and selectivity. Oligopyrimidine PNA can displace one strand of dsDNA by forming PNA(2):DNA triplexes of very high stability. Many PNA analogs have been described in recent years, in particular, chiral PNA analogs. In the present article the results obtained recently using PNA derived from N-aminoethylamino acids 7 are illustrated. In particular, the dependence of optical purity on synthetic methodologies and a rationale for the observed effects of chirality on DNA binding ability is proposed. Chirality as a tool for improving sequence selectivity is also described. PNA analogs derived from D- or L-ornithine 8 were also found to be subjected to epimerization during solid phase synthesis. Modification of the coupling conditions or the use of a submonomeric strategy greatly reduced epimerization. The optically pure oligothymine PNAs 8 were found to bind to RNA by forming triplexes of unusual CD spectra. The melting curves of these adducts presented two transitions, suggesting a conformational change followed by melting at high temperature.

Chemistry↗

PubMedDate not supplied

Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor.

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.

Cystic Fibrosis↗