38 results on '"Sforza, Stefano"'
Search Results
2. Chiral PNAs with constrained open-chain backbones.
- Author
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Corradini R, Tedeschi T, Sforza S, and Marchelli R
- Subjects
- Biological Transport, Chromatography, Gas, DNA metabolism, Fluorenes chemistry, Models, Molecular, Nucleic Acid Conformation, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids metabolism, Permeability, RNA metabolism, Solid-Phase Synthesis Techniques, Stereoisomerism, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2014
- Full Text
- View/download PDF
3. PNA-NLS conjugates as single-molecular activators of target sites in double-stranded DNA for site-selective scission.
- Author
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Aiba Y, Hamano Y, Kameshima W, Araki Y, Wada T, Accetta A, Sforza S, Corradini R, Marchelli R, and Komiyama M
- Subjects
- Base Pair Mismatch, Base Sequence, Cerium chemistry, DNA genetics, Edetic Acid chemistry, Humans, Spectrometry, Fluorescence, DNA chemistry, Nuclear Localization Signals, Peptide Nucleic Acids chemistry
- Abstract
Artificial DNA cutters have been developed by us in our previous studies by combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with Ce(IV)-EDTA-promoted hydrolysis. The pcPNAs have two modified nucleobases (2,6-diaminopurine and 2-thiouracil) instead of conventional A and T, and can invade double-stranded DNA to activate the target site for the scission. This system has been applied to site-selective scissions of plasmid, λ-phage, E. coli genomic DNA, and human genomic DNA. Here, we have reported a still simpler and more convenient DNA cutter obtained by conjugating peptide nucleic acid (PNA) with a nuclear localization signal (NLS) peptide. This new DNA cutter requires only one PNA strand (instead of two) bearing conventional (non-pseudo-complementary) nucleobases. This PNA-NLS conjugate effectively activated the target site in double-stranded DNA and induced site-selective scission by Ce(IV)-EDTA. The complex formation between the conjugate and DNA was concretely evidenced by spectroscopic results based on time-resolved fluorescence. The target scission site of this new system was straightforwardly determined by the Watson-Crick base pairing rule, and mismatched sequences were clearly discriminated. Importantly, even highly GC-rich regions, which are difficult to be targeted by a previous strategy using pcPNA, were successfully targeted. All these features of the present DNA cutter make it promising for various future applications.
- Published
- 2013
- Full Text
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4. Cellular uptakes, biostabilities and anti-miR-210 activities of chiral arginine-PNAs in leukaemic K562 cells.
- Author
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Manicardi A, Fabbri E, Tedeschi T, Sforza S, Bianchi N, Brognara E, Gambari R, Marchelli R, and Corradini R
- Subjects
- Base Sequence, Biological Transport, Cell Differentiation drug effects, DNA chemistry, Erythroid Cells cytology, Erythroid Cells drug effects, Humans, K562 Cells, Nucleic Acid Denaturation, Nucleic Acid Hybridization, Peptide Nucleic Acids genetics, Peptide Nucleic Acids pharmacology, RNA chemistry, Stereoisomerism, Transition Temperature, Arginine, Leukemia pathology, MicroRNAs genetics, Peptide Nucleic Acids chemistry, Peptide Nucleic Acids metabolism
- Abstract
A series of 18-mer peptide nucleic acids (PNAs) targeted against micro-RNA miR-210 was synthesised and tested in a cellular system. Unmodified PNAs, R(8) -conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2-modified (R) or C5-modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 M urea was used to assess differences between the different structures. FACS analysis and qRT-PCR on K562 chronic myelogenous leukaemic cells indicated that arginine-conjugated and backbone-modified PNAs display good cellular uptake, with best performances for the C2-modified series. Resistance to enzymatic degradation was found to be higher for the backbone-modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR-210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin-treated cells. Interestingly, the anti-miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone-modified PNAs as anti-miR agents. The results clearly indicate that backbone-modified PNAs are good candidates for the development of very efficient drugs based on anti-miR activity, due to their enhanced bioavailabilities, and that overall anti-miR performance is a combination of cellular uptake and RNA binding., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
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5. Letter from the editors.
- Author
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Corradini R and Sforza S
- Subjects
- Chemistry Techniques, Synthetic, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids chemistry, Peptide Nucleic Acids metabolism
- Published
- 2012
- Full Text
- View/download PDF
6. PNA bearing 5-azidomethyluracil: a novel approach for solid and solution phase modification.
- Author
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Manicardi A, Accetta A, Tedeschi T, Sforza S, Marchelli R, and Corradini R
- Subjects
- Azides chemistry, Base Sequence, Click Chemistry, Drug Design, Peptide Nucleic Acids genetics, Solutions, Thymine chemistry, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids chemistry, Thymine analogs & derivatives, Uracil chemistry
- Abstract
Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition.
- Published
- 2012
- Full Text
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7. Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays.
- Author
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Rossi S, Calabretta A, Tedeschi T, Sforza S, Arcioni S, Baldoni L, Corradini R, and Marchelli R
- Subjects
- Corylus chemistry, Corylus genetics, Food Contamination analysis, Olive Oil, Peptide Nucleic Acids genetics, Polymorphism, Single Nucleotide, Substrate Specificity, DNA, Plant analysis, DNA, Plant genetics, Olea genetics, Oligonucleotide Array Sequence Analysis methods, Peptide Nucleic Acids analysis, Plant Leaves genetics, Plant Oils chemistry
- Abstract
PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.
- Published
- 2012
- Full Text
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8. Modulation of the biological activity of microRNA-210 with peptide nucleic acids (PNAs).
- Author
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Fabbri E, Manicardi A, Tedeschi T, Sforza S, Bianchi N, Brognara E, Finotti A, Breveglieri G, Borgatti M, Corradini R, Marchelli R, and Gambari R
- Subjects
- Base Sequence, Cell Line, Tumor, Humans, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids pharmacology, Peptides chemistry, Phase Transition, Ultraviolet Rays, gamma-Globins genetics, gamma-Globins metabolism, MicroRNAs metabolism, Peptide Nucleic Acids chemistry
- Abstract
Herein we describe the activity of a peptide nucleic acid (PNA) that targets microRNA-210 (miR-210), which is associated with hypoxia and is modulated during erythroid differentiation. PNAs directed against miR-210 were designed to bind with high affinity to the target RNA strand and to undergo efficient uptake in target cells. A polyarginine-PNA conjugate directed against miR-210 (Rpep-PNA-a210) showed both very high affinity for RNA and efficient uptake into target cells without the need for transfection reagents. An unmodified PNA of the same sequence displayed the ability to bind RNA, but cellular uptake was very poor. Consistent with this, only Rpep-PNA-a210 strongly inhibited miR-210 activity, as evaluated by assays on undifferentiated K562 cells and on cells treated with mithramycin, which was found to induce erythroid differentiation and miR-210 overexpression. Targeting miR-210 by Rpep-PNA-a210 resulted in: 1) a decrease in miR-210 levels as measured by RT-PCR, 2) up-regulation of raptor mRNA, 3) a decrease in γ-globin mRNA, and 4) decreased expression of differentiated functions (i.e., proportion of benzidine-positive cells, content of embryo-fetal hemoglobins). The efficient delivery of anti-miR PNAs through a suitable peptide carrier (Rpep-PNA-a210) leads to the inhibition of miR-210 activity, altering the expression of miR-210-regulated erythroid functions., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2011
- Full Text
- View/download PDF
9. miRNA therapeutics: delivery and biological activity of peptide nucleic acids targeting miRNAs.
- Author
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Fabbri E, Brognara E, Borgatti M, Lampronti I, Finotti A, Bianchi N, Sforza S, Tedeschi T, Manicardi A, Marchelli R, Corradini R, and Gambari R
- Subjects
- Humans, Peptide Nucleic Acids administration & dosage, Drug Delivery Systems methods, Drug Discovery methods, Gene Expression Regulation physiology, MicroRNAs drug effects, MicroRNAs metabolism, Models, Biological, Peptide Nucleic Acids metabolism, Peptide Nucleic Acids pharmacology
- Abstract
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.
- Published
- 2011
- Full Text
- View/download PDF
10. A PNA microarray for tomato genotyping.
- Author
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Tedeschi T, Calabretta A, Bencivenni M, Manicardi A, Corrado G, Caramante M, Corradini R, Rao R, Sforza S, and Marchelli R
- Subjects
- Base Sequence, Genotype, Molecular Probes chemical synthesis, Molecular Structure, Peptide Nucleic Acids chemical synthesis, Polymorphism, Single Nucleotide, Transition Temperature, Solanum lycopersicum genetics, Molecular Probes chemistry, Oligonucleotide Array Sequence Analysis methods, Peptide Nucleic Acids chemistry
- Abstract
The design and development of a PNA microarray designed for the simultaneous identification of several SNPs characteristic of seven different tomato varieties is described. Highly selective arginine-based monomer containing PNAs (Arg-PNAs) have been used in order to obtain very selective probes. Seven modified PNA probes were synthesised and their binding properties in solution were studied. PNA-microarrays based on these probes were prepared and applied to SNP discrimination in model experiments using oligonucleotide mixtures simulating the different sequences of the seven tomato varieties. The strength and the limitations of such a system for SNP recognition are thoroughly discussed.
- Published
- 2011
- Full Text
- View/download PDF
11. Real time RNA transcription monitoring by Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes: norovirus detection.
- Author
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Tonelli A, Tedeschi T, Germini A, Sforza S, Corradini R, Medici MC, Chezzi C, and Marchelli R
- Subjects
- Base Sequence, Caliciviridae Infections diagnosis, Caliciviridae Infections virology, Feces virology, Gastroenteritis diagnosis, Gastroenteritis virology, Humans, Models, Genetic, Molecular Sequence Data, Molecular Structure, Norovirus genetics, Nucleic Acid Amplification Techniques methods, Nucleic Acid Conformation, Nucleic Acid Probes chemistry, Peptide Nucleic Acids chemistry, Reproducibility of Results, Sensitivity and Specificity, Transition Temperature, Viral Proteins genetics, Benzothiazoles chemistry, Nucleic Acid Probes genetics, Peptide Nucleic Acids genetics, Quinolines chemistry, RNA, Viral genetics, Transcription, Genetic
- Abstract
Thiazole Orange (TO)-conjugated Peptide Nucleic Acid (PNA) probes have been reported as a valuable strategy for DNA analysis; however, no investigations targeting RNA molecules and no comparisons between different derivatization approaches have been reported so far. In this work, two TO-conjugated PNAs for genogroup II noroviruses (NoV GII) detection were designed and synthesized. Both the probes target the most conserved stretch of nucleotides identified in the open reading frame 1-2 (ORF1-ORF2) junction region and differ for the dye conjugation strategy: one PNA is end-labelled with the TO molecule tethered by a linker; the other probe bears the TO molecule directly linked to the PNA backbone, replacing a conventional nucleobase. The spectroscopic properties of the two PNA probes were studied and their applicability to NoVs detection, using an isothermal assay, was investigated. Both probes showed good specificity and high fluorescence enhancement upon hybridization, especially targeting RNA molecules. Moreover, the two probes were successfully employed for NoVs detection from stool specimens in an isothermal-based amplification assay targeting RNA 'amplicons'. The probes showed to be specific even in the presence of high concentrations of non-target RNA.
- Published
- 2011
- Full Text
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12. Patterning of peptide nucleic acids using reactive microcontact printing.
- Author
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Calabretta A, Wasserberg D, Posthuma-Trumpie GA, Subramaniam V, van Amerongen A, Corradini R, Tedeschi T, Sforza S, Reinhoudt DN, Marchelli R, Huskens J, and Jonkheijm P
- Subjects
- Microscopy, Fluorescence, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2011
- Full Text
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13. Food analysis and food authentication by peptide nucleic acid (PNA)-based technologies.
- Author
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Sforza S, Corradini R, Tedeschi T, and Marchelli R
- Subjects
- Allergens chemistry, Allergens immunology, DNA chemistry, DNA metabolism, Food Contamination analysis, Metal Nanoparticles chemistry, Organisms, Genetically Modified genetics, Organisms, Genetically Modified microbiology, Food Analysis, Peptide Nucleic Acids chemistry
- Abstract
This tutorial review will address the issue of DNA determination in food by using Peptide Nucleic Acid (PNA) probes with different technological platforms, with a particular emphasis on the applications devoted to food authentication. After an introduction aimed at describing PNAs structure, binding properties and their use as genetic probes, the review will then focus specifically on the use of PNAs in the field of food analysis. In particular, the following issues will be considered: detection of genetically modified organisms (GMOs), of hidden allergens, of microbial pathogens and determination of ingredient authenticity. Finally, the future perspectives for the use of PNAs in food analysis will be briefly discussed according to the most recent developments.
- Published
- 2011
- Full Text
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14. Peptide nucleic acids with a structurally biased backbone. Updated review and emerging challenges.
- Author
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Corradini R, Sforza S, Tedeschi T, Totsingan F, Manicardi A, and Marchelli R
- Subjects
- Animals, Gene Expression Regulation, Humans, Nucleic Acid Conformation, Peptide Nucleic Acids pharmacology, RNA metabolism, Peptide Nucleic Acids chemistry, Peptide Nucleic Acids therapeutic use
- Abstract
Peptide nucleic acids (PNAs) are polyamidic oligonucleotide analogues which have been described for the first time almost twenty years ago and were immediately found to be excellent tool in binding DNA and RNA for diagnostics and gene regulation. Their use as therapeutic agents have been proposed since early studies and recent advancements in cellular delivery systems and in the so called anti-gene strategy make them good candidates for drug development. The search for new chemical modification of PNAs is a very active field of research and new structures are continuously proposed. This review focuses on the modification of the PNA backbone, and their possible use in medicinal chemistry with an update of this topics in view of emerging new trends and opening of new possibilities In particular two classes of structurally biased PNAs are described in details: i) PNAs with acyclic structures and their helical preference, which is regulated by stereochemistry and ii) cyclic PNAs with preorganized structures, whose performances depend both on stereochemistry and on conformational constraints. The properties of these compounds are discussed in terms of affinity for nucleic acids, and several examples of their use in cellular or animal systems are presented , with exciting new fields of research such as microRNA (miR) targeting and gene repair.
- Published
- 2011
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15. Control of helical handedness in DNA and PNA nanostructures.
- Author
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Corradini R, Tedeschi T, Sforza S, Green MM, and Marchelli R
- Subjects
- Base Pairing, Base Sequence, Models, Molecular, Nucleic Acid Conformation, DNA, B-Form chemistry, DNA, Z-Form chemistry, Nanostructures chemistry, Nanotechnology methods, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2011
- Full Text
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16. Affinity and selectivity of C2- and C5-substituted "chiral-box" PNA in solution and on microarrays.
- Author
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Manicardi A, Calabretta A, Bencivenni M, Tedeschi T, Sforza S, Corradini R, and Marchelli R
- Subjects
- Circular Dichroism, Models, Molecular, Molecular Structure, Solutions, Stereoisomerism, Microarray Analysis instrumentation, Microarray Analysis methods, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids chemistry
- Abstract
Two peptide nucleic acids (PNAs) containing three adjacent modified chiral monomers (chiral box) were synthesized. The chiral monomers contained either a C2- or a C5-modified backbone, synthesized starting from D- and L-arginine, respectively (2D- and 5L-PNA). The C2-modified chiral PNA was synthesized using a submonomeric strategy to avoid epimerization during solid-phase synthesis, whereas for the C5-derivative, the monomers were first obtained and then used in solid-phase synthesis. The melting temperature of these PNA duplexes formed with the full-match or with single-mismatch DNA were measured both by UV and by CD spectroscopy and compared with the unmodified PNA. The 5L-chiral-box-PNA showed the highest T(m) with full-match DNA, whereas the 2D-chiral-box-PNA showed the highest sequence selectivity. The PNA were spotted on microarray slides and then hybridized with Cy5-labeled full match and mismatched oligonucleotides. The results obtained showed a signal intensity in the order achiral >2D-chiral box >5L-chiral box, whereas the full-match/mismatch selectivity was higher for the 2D chiral box PNA., (© 2010 Wiley-Liss, Inc.)
- Published
- 2010
- Full Text
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17. SSB-assisted duplex invasion of preorganized PNA into double-stranded DNA.
- Author
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Ishizuka T, Tedeschi T, Corradini R, Komiyama M, Sforza S, and Marchelli R
- Subjects
- Base Sequence, Circular Dichroism, DNA Primers, Electrophoresis, Polyacrylamide Gel, DNA chemistry, Peptide Nucleic Acids chemistry
- Published
- 2009
- Full Text
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18. Arginine-based PNA microarrays for APOE genotyping.
- Author
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Calabretta A, Tedeschi T, Di Cola G, Corradini R, Sforza S, and Marchelli R
- Subjects
- Humans, Molecular Structure, Polymerase Chain Reaction, Polymorphism, Single Nucleotide genetics, Transition Temperature, Apolipoproteins E genetics, Arginine chemistry, Genotype, Peptide Nucleic Acids genetics
- Abstract
Four modified PNAs containing one chiral monomer bearing two arginine-derived side chains, with the correct configuration for specific and stable DNA binding, were synthesized, complementary to two DNA tracts in the APOE gene containing SNPs related to the insurgence of Alzheimer's disease. PNA binding performances were first tested in solution against complementary and mismatched oligonucleotides by measuring melting temperatures, and showed high specificity in SNP recognition. In order to set up a new diagnostic platform for APOE genotyping, PNA microarrays were then developed with the synthesized modified PNAs. PNA probe deposition protocols on microarrays were optimized in order to minimize cross-contamination due to carry over. The microarrays obtained by arginine-based PNA deposition were incubated with complementary and mismatched oligonucleotides, showing excellent mismatch recognition on the microarray platform. The specificity of the microarrays was finally tested with oligonucleotide mixtures simulating the real genotype profiles. Six different hybridisation patterns related to six different genotypes in the APOE gene were found to be clearly distinct in microarray experiments, demonstrating the potential of this approach for highly specific genetic analysis.
- Published
- 2009
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19. Highly selective single nucleotide polymorphism recognition by a chiral (5S) PNA beacon.
- Author
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Totsingan F, Tedeschi T, Sforza S, Corradini R, and Marchelli R
- Subjects
- Base Pair Mismatch, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, DNA metabolism, Fluorescence, Nucleic Acid Denaturation, Peptide Nucleic Acids chemical synthesis, Spectrophotometry, Ultraviolet, Stereoisomerism, Substrate Specificity, Transition Temperature, DNA analysis, DNA genetics, Peptide Nucleic Acids chemistry, Peptide Nucleic Acids metabolism, Polymorphism, Single Nucleotide
- Abstract
A chiral peptide nucleic acid (PNA) beacon containing a C-5 modified monomer based on L-lysine was synthesized. The terminal amino group of the lysine side chain was linked to a spacer for future applications on surfaces. The PNA beacon bears a carboxyfluorescein fluorophore and a dabcyl quencher at opposite ends. The DNA binding properties were compared with those of a homologous PNA beacon containing only achiral monomers. Both beacons underwent a fluorescence increase in the presence of complementary DNA, with higher efficiency and higher selectivity (evaluated using single mismatched DNA sequences) observed for the chiral monomer containing PNA. Ion exchange (IE) HPLC with fluorimetric detection was used in combination with the beacon for the selective detection of complementary DNA. A fluorescent peak corresponding to the PNA beacon:DNA duplex was observed at a very low detection limit (1 nM). The discriminating capacity of the chiral PNA beacon for a single mismatch was found to be superior to those observed with the unmodified one, thus confirming the potency of chirality for increasing the affinity and specificity of DNA recognition., ((c) 2008 Wiley-Liss, Inc.)
- Published
- 2009
- Full Text
- View/download PDF
20. Promotion of strand invasion by utilizing entropically-favored PNA.
- Author
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Ishizuka T, Sforza S, and Komiyama M
- Subjects
- Entropy, Thermodynamics, DNA chemistry, Peptide Nucleic Acids chemistry
- Abstract
Invasion of peptide nucleic acid (PNA) is important to target a specific site in double-stranded DNA. However, it needs improvement in many ways such as sequence versatility and invasion efficiency. It is here reported that one 10-mer strand of alpha-lysine substituted peptide nucleic acid (PNA) efficiently invades double-stranded DNA (dsDNA) in the presence of single-strand binding protein (SSB) via Watson-Crick rule. According to the thermodynamic parameters in this study, highly efficient invasion was achieved by a favorable entropy change. The importance of preorganization of PNAs for efficient strand invasion is indicated.
- Published
- 2009
- Full Text
- View/download PDF
21. Label-free selective DNA detection with high mismatch recognition by PNA beacons and ion exchange HPLC.
- Author
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Totsingan F, Rossi S, Corradini R, Tedeschi T, Sforza S, Juris A, Scaravelli E, and Marchelli R
- Subjects
- Base Pair Mismatch, Base Sequence, Chromatography, High Pressure Liquid, DNA analysis, Ion Exchange Resins chemistry, Molecular Sequence Data, Molecular Structure, DNA chemistry, Peptide Nucleic Acids chemistry
- Abstract
Two 11mer peptide nucleic acid (PNA) beacons were synthesized and tested for the detection of full-matched or single mismatched DNA. Fluorescent measurements carried out in solution showed only partial discrimination of the mismatched sequence, while using anion-exchange HPLC, in combination with fluorimetric detection, allowed DNA analysis to be performed with high sensitivity and extremely high sequence selectivity. Up to >90 : 1 signal discrimination in the presence of one single mismatched base was observed. The analysis was tested on both short and long DNA oligomers. Detection of DNA obtained from PCR amplification was also performed allowing the selective detection of the target sequence in complex mixtures. Label free detection of the DNA with high sequence selectivity is therefore possible using the present approach.
- Published
- 2008
- Full Text
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22. Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences.
- Author
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Ishizuka T, Yoshida J, Yamamoto Y, Sumaoka J, Tedeschi T, Corradini R, Sforza S, and Komiyama M
- Subjects
- 2-Aminopurine analogs & derivatives, 2-Aminopurine chemistry, AT Rich Sequence, Base Pair Mismatch, Base Sequence, GC Rich Sequence, Static Electricity, Stereoisomerism, Thiouracil chemistry, DNA chemistry, Peptide Nucleic Acids chemistry
- Abstract
Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this 'double-duplex invasion', a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the alpha-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsen's PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G-C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.
- Published
- 2008
- Full Text
- View/download PDF
23. Circular dichroism study of DNA binding by a potential anticancer peptide nucleic acid targeted against the MYCN oncogene.
- Author
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Faccini A, Tortori A, Tedeschi T, Sforza S, Tonelli R, Pession A, Corradini R, and Marchelli R
- Subjects
- Base Sequence, Binding, Competitive, Circular Dichroism, DNA chemistry, DNA, Antisense genetics, Models, Molecular, Molecular Conformation, Nuclear Localization Signals genetics, Peptide Nucleic Acids chemistry, Spectrophotometry, Ultraviolet, Stereoisomerism, DNA genetics, DNA metabolism, Genes, myc drug effects, Peptide Nucleic Acids metabolism, Peptide Nucleic Acids pharmacology
- Abstract
The interaction with DNA of a peptide nucleic acid (PNA) oligomer (16nt) conjugated with a nuclear localization signal (NLS) peptide, which was previously found to be able to inhibit tumor cell proliferation through block of transcription of the MYCN oncogene, was studied by UV and CD spectroscopy. While data obtained by UV were not conclusive, the use of circular dichroism gave clear-cut evidence of the formation of a PNA:DNA duplex of exceptionally high stability (Tm >or= 90 degrees C). Using the same approach, the effect of mutations on DNA:PNA stability was evaluated, and was found in accordance with that expected for a Watson-Crick interaction. The role of the NLS peptide was evaluated by using a PNA lacking of this part, which gave rise to less stable PNA:DNA duplexes. Finally, a competition experiment carried out with a 26mer dsDNA, containing the target 16mer sequence in its middle region, in the presence of PNA-NLS gave evidence for the formation of a ternary complex at 25 degrees , while at higher temperature, the PNA:DNA duplex and the displaced homologous DNA strand were detected. The present results support the possibility of an analogous mechanism of action of this antitumor PNA in vivo., ((c) 2007 Wiley-Liss, Inc.)
- Published
- 2008
- Full Text
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24. Fast and easy colorimetric tests for single mismatch recognition by PNA-DNA duplexes with the diethylthiadicarbocyanine dye and succinyl-beta-cyclodextrin.
- Author
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Tedeschi T, Sforza S, Ye S, Corradini R, Dossena A, Komiyama M, and Marchelli R
- Subjects
- Apolipoproteins E genetics, Base Pair Mismatch, Base Sequence, Coloring Agents, DNA genetics, DNA Primers genetics, Dithiazanine, Fluorescent Dyes, Humans, In Vitro Techniques, Macromolecular Substances chemistry, Molecular Sequence Data, Point Mutation, Polymorphism, Single Nucleotide, Solvents, Spectrophotometry, beta-Cyclodextrins chemistry, Colorimetry methods, DNA chemistry, Peptide Nucleic Acids chemistry
- Abstract
The 3,3'-diethylthiacarbocyanine (DiSC(2)(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-beta-cyclodextrin (Succ-beta-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 DiSC(2)(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.
- Published
- 2007
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- View/download PDF
25. Peptide nucleic acids with a structurally biased backbone: effects of conformational constraints and stereochemistry.
- Author
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Corradini R, Sforza S, Tedeschi T, Totsingan F, and Marchelli R
- Subjects
- Animals, Cyclization, DNA chemistry, Humans, Molecular Conformation, Stereoisomerism, Peptide Nucleic Acids chemistry
- Abstract
Peptide nucleic acids (PNAs) are polyamidic oligonucleotide analogs which have been described for the first time fifteen years ago and were immediately found to be excellent tools in binding DNA and RNA for diagnostics and gene regulation. Their use as therapeutic agents have been proposed since early studies and recent advancements in cellular delivery systems, and in the so called anti-gene strategy, makes them good candidates for drug development. The search for new chemical modification of PNAs is a very active field of research and new structures are continuously proposed. This review focuses on the recent advancements obtained by the modification of the PNA backbone, and their possible use in medicinal chemistry. In particular two classes of structurally biased PNAs are described in details: i) PNAs with acyclic structures and their helical preference, which is regulated by stereochemistry and ii) cyclic PNAs with preorganized structures, whose performances depend both on stereochemistry and on conformational constraints. The properties of these compounds are discussed in terms of affinity for nucleic acids, and several recent examples of their use in cellular or animal systems are presented.
- Published
- 2007
- Full Text
- View/download PDF
26. A new concept in double duplex DNA invasion by chiral PNAs which simultaneously depress PNA-PNA and improve PNA-DNA duplex stability.
- Author
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Sforza S, Tedeschi T, Corradini R, and Marchelli R
- Subjects
- Lysine chemistry, Nucleic Acid Denaturation, Peptide Nucleic Acids chemical synthesis, Stereoisomerism, DNA chemistry, Peptide Nucleic Acids chemistry
- Abstract
By using suitably designed chiral, lysine-based PNAs (chPNAs), the stability of complementary chPNAchPNA duplexes can be depressed, maintaining or improving at the same time the stability of the corresponding PNA-DNA duplexes. This approach could be used in order to develop chPNAs able to perform double duplex DNA invasion.
- Published
- 2007
- Full Text
- View/download PDF
27. Highly efficient strand invasion by peptide nucleic acid bearing optically pure lysine residues in its backbone.
- Author
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Yamamoto Y, Yoshida J, Tedeschi T, Corradini R, Sforza S, and Komiyama M
- Subjects
- 2-Aminopurine analogs & derivatives, 2-Aminopurine chemistry, Electrophoretic Mobility Shift Assay, GC Rich Sequence, Isomerism, Lysine chemistry, Static Electricity, Thiouracil chemistry, DNA chemistry, Lysine analogs & derivatives, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2006
- Full Text
- View/download PDF
28. Unconventional method based on circular dichroism to detect peanut DNA in food by means of a PNA probe and a cyanine dye.
- Author
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Sforza S, Scaravelli E, Corradini R, and Marchelli R
- Subjects
- Circular Dichroism, DNA Probes analysis, DNA Probes genetics, DNA, Plant genetics, Molecular Structure, Peptide Nucleic Acids chemistry, Peptide Nucleic Acids genetics, Arachis genetics, Carbocyanines analysis, DNA, Plant analysis, Food Analysis methods, Peptide Nucleic Acids analysis
- Abstract
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)., (Copyright 2005 Wiley-Liss, Inc.)
- Published
- 2005
- Full Text
- View/download PDF
29. 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.
- Author
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Tedeschi T, Chiari M, Galaverna G, Sforza S, Cretich M, Corradini R, and Marchelli R
- Subjects
- Cystic Fibrosis Transmembrane Conductance Regulator genetics, DNA Probes, Electrophoresis, Capillary, Humans, Lysine chemistry, Nucleic Acid Hybridization methods, Polymerase Chain Reaction, Polymorphism, Single Nucleotide, Stereoisomerism, Cystic Fibrosis genetics, Peptide Nucleic Acids chemistry, Point Mutation genetics
- Abstract
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.
- Published
- 2005
- Full Text
- View/download PDF
30. Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis.
- Author
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Tonelli R, Purgato S, Camerin C, Fronza R, Bologna F, Alboresi S, Franzoni M, Corradini R, Sforza S, Faccini A, Shohet JM, Marchelli R, and Pession A
- Subjects
- Humans, Tumor Cells, Cultured, Apoptosis drug effects, Gene Expression Regulation, Neoplastic drug effects, Neuroblastoma drug therapy, Neuroblastoma metabolism, Peptide Nucleic Acids therapeutic use, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Proto-Oncogene Proteins c-myc metabolism
- Abstract
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.
- Published
- 2005
- Full Text
- View/download PDF
31. Lysine-based peptide nucleic acids (PNAs) with strong chiral constraint: control of helix handedness and DNA binding by chirality.
- Author
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Tedeschi T, Sforza S, Dossena A, Corradini R, and Marchelli R
- Subjects
- Base Pair Mismatch, Base Sequence, DNA chemistry, Drug Stability, Lysine chemistry, Nucleic Acid Conformation, Peptide Nucleic Acids chemical synthesis, Spectrometry, Mass, Electrospray Ionization, Spectrophotometry, Ultraviolet, Stereoisomerism, DNA metabolism, Peptide Nucleic Acids chemistry, Peptide Nucleic Acids metabolism
- Abstract
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., (2005 Wiley-Liss, Inc.)
- Published
- 2005
- Full Text
- View/download PDF
32. 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.
- Author
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Pession A, Tonelli R, Fronza R, Sciamanna E, Corradini R, Sforza S, Tedeschi T, Marchelli R, Montanaro L, Camerin C, Franzoni M, and Paolucci G
- Subjects
- Apoptosis, Base Sequence, Blotting, Western, Cell Cycle, Cell Differentiation, Cell Division, Cell Line, Tumor, DNA Primers pharmacology, Flow Cytometry, G1 Phase, Humans, Microscopy, Fluorescence, Molecular Sequence Data, Neurons pathology, Point Mutation, Reverse Transcriptase Polymerase Chain Reaction, Neuroblastoma drug therapy, Neuroblastoma metabolism, Peptide Nucleic Acids therapeutic use, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Proto-Oncogene Proteins c-myc biosynthesis
- Abstract
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.
- Published
- 2004
33. Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor.
- Author
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Corradini R, Feriotto G, Sforza S, Marchelli R, and Gambari R
- Subjects
- Humans, Lysine genetics, Nucleic Acid Hybridization, Peptide Nucleic Acids chemical synthesis, Peptide Nucleic Acids chemistry, Sensitivity and Specificity, Transition Temperature, Cystic Fibrosis genetics, Mutation, Missense genetics, Peptide Nucleic Acids analysis, Peptide Nucleic Acids genetics, Point Mutation genetics, Surface Plasmon Resonance instrumentation, Surface Plasmon Resonance methods
- Abstract
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.)
- Published
- 2004
- Full Text
- View/download PDF
34. Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine-based chiral PNA-DNA duplex.
- Author
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Menchise V, De Simone G, Tedeschi T, Corradini R, Sforza S, Marchelli R, Capasso D, Saviano M, and Pedone C
- Subjects
- Base Sequence, Crystallography, X-Ray, Macromolecular Substances, Models, Molecular, Molecular Structure, Nucleic Acid Heteroduplexes chemistry, Peptide Nucleic Acids chemical synthesis, Static Electricity, Stereoisomerism, DNA chemistry, Lysine chemistry, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
35. Enantiomeric separation of chiral peptide nucleic acid monomers by capillary electrophoresis with charged cyclodextrins.
- Author
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Galaverna G, Sforza S, Tedeschi T, Corradini R, Dossena A, and Marchelli R
- Subjects
- Hydrogen-Ion Concentration, Static Electricity, Stereoisomerism, Cyclodextrins chemistry, Electrophoresis, Capillary methods, Peptide Nucleic Acids isolation & purification
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
36. Direction control in DNA binding of chiral D-lysine-based peptide nucleic acid (PNA) probed by electrospray mass spectrometry.
- Author
-
Sforza S, Tedeschi T, Corradini R, Dossena A, and Marchelli R
- Subjects
- Molecular Structure, Spectrometry, Mass, Electrospray Ionization, Stereoisomerism, DNA chemistry, Lysine chemistry, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2003
- Full Text
- View/download PDF
37. Role of chirality and optical purity in nucleic acid recognition by PNA and PNA analogs.
- Author
-
Sforza S, Galaverna G, Dossena A, Corradini R, and Marchelli R
- Subjects
- Chemistry methods, Circular Dichroism, Gas Chromatography-Mass Spectrometry, Models, Chemical, RNA chemistry, RNA metabolism, Spectrophotometry, Stereoisomerism, Temperature, Thermodynamics, Ultraviolet Rays, DNA chemistry, Nucleic Acids chemistry, Peptide Nucleic Acids chemistry
- Abstract
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., (Copyright 2002 Wiley-Liss, Inc.)
- Published
- 2002
- Full Text
- View/download PDF
38. Crystallization and preliminary X-ray diffraction studies of a D-lysine-based chiral PNA-DNA duplex.
- Author
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Menchise V, De Simone G, Corradini R, Sforza S, Sorrentino N, Romanelli A, Saviano M, and Pedone C
- Subjects
- Crystallization, Crystallography, X-Ray, Polyethylene Glycols chemistry, Protein Conformation, DNA chemistry, Lysine chemistry, Nucleic Acid Conformation, Peptide Nucleic Acids chemistry
- Abstract
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.
- Published
- 2002
- Full Text
- View/download PDF
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