Parallel synthesis of PNA-peptide conjugate libraries

An optimized semi-automatic protocol for parallel synthesis of up to 96 peptide nucleic acids (PNA) or PNA-peptide conjugates using Boc-protection strategy has been developed using a robotic system. The approach is illustrated by synthesizing PNA and PNA-peptide libraries varying between 15 and 27 amino acid units. The peptides (NLS (nuclear localization signal) or Tat-peptide) were attached to N-terminus of the PNA. The method was found to be far superior to that based on the SPOT/Fmoc protocol by which PNA oligomers are synthesized on a modified cellulose membrane. On a 0.5 micromole scale the method typically yielded 2 mg product of 90% purity by HPLC/MALDI-TOF analysis. This approach is suitable for screening of a large number of PNA and/or peptide sequences for biochemical and biological studies.     

Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.

     

Cyclopropane PNA: observable triplex melting in a PNA constrained with a 3-membered ring

The first peptide nucleic acid (PNA) with a cyclopropane in the backbone has been synthesized, and the effects of the ring on DNA/RNA binding properties of the PNA have been examined. Well-defined triplex to duplex melting transitions of PNA₂ DNA complexes is clearly observed by variable temperature UV absorbance with the cyclopropane-constrained PNA.     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Synthesis and characterization of novel poly(1-naphthylamine)/zinc oxide nanocomposites: Application in catalytic degradation of methylene blue dye

The organic–inorganic poly(1-naphthylamine)/ZnO (PNA/ZnO) nanocomposites were synthesized by in situ chemical oxidative polymerization of 1-naphthylamine monomer and ZnO nanomaterials for the photocatalytic degradation of methylene blue (MB) dye under visible light. The surface properties of PNA molecules were altered by adding the ZnO nanomaterials. The crystalline and the optical properties of PNA/ZnO nanocomposites were improved with the increased contents of ZnO nanomaterials in PNA molecules due to the effective interaction of PNA to the surface of ZnO nanomaterials. The prepared PNA/ZnO nanocomposites presented moderate photocatalytic MB dye degradation of ~22% under visible light. The occurrence of dye degradation under visible light might attribute to high-charge separation of ē-h⁺ pairs at the interfaces of PNA and ZnO nanomaterials in its excited state under light irradiation.     

Electroactive chitosan nanoparticles for the detection of single-nucleotide polymorphisms using peptide nucleic acids

Here we report an electrochemical biosensor that would allow for simple and rapid analysis of nucleic acids in combination with nuclease activity on nucleic acids and electroactive bionanoparticles. The detection of single-nucleotide polymorphisms (SNPs) using PNA probes takes advantage of the significant structural and physicochemical differences between the full hybrids and SNPs in PNA/DNA and DNA/DNA duplexes. Ferrocene-conjugated chitosan nanoparticles (Chi-Fc) were used as the electroactive indicator of hybridization. Chi-Fc had no affinity towards the neutral PNA probe immobilized on a gold electrode (AuE) surface. When the PNA probe on the electrode surface hybridized with a full-complementary target DNA, Chi-Fc electrostatically attached to the negatively-charged phosphate backbone of DNA on the surface and gave rise to a high electrochemical oxidation signal from ferrocene at ∼0.30 V. Exposing the surface to a single-stranded DNA specific nuclease, Nuclease S1, was found to be very effective for removing the nonspecifically adsorbed SNP DNA. An SNP in the target DNA to PNA made it susceptible to the enzymatic digestion. After the enzymatic digestion and subsequent exposure to Chi-Fc, the presence of SNPs was determined by monitoring the changes in the electrical current response of Chi-Fc. The method provided a detection limit of 1 fM (S/N = 3) for the target DNA oligonucleotide. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism (GMO) in standard Roundup Ready soybean samples. PNA-mediated PCR amplification of real DNA samples was performed to detect SNPs related to alcolohol dehydrogenase (ALDH). Chitosan nanoparticles are promising biometarials for various analytical and pharmaceutical applications. Figure The electrochemical method for SNP detection using PNA probes and chitosan nanoparticles takes advantage of the significant structural and physicochemical differences between PNA/DNA and DNA/DNA duplexes. Single-stranded DNA specific enzymes selectively choose these SNP sites and hydrolyze the DNA molecules on gold electrode (AuE) surface. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cyanuryl peptide nucleic acid: synthesis and DNA complexation properties

The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-aminoethyl)-N′-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids when the cyanuryl unit was located in the middle of the PNA oligomer.     

Synthesis of thiol-modified peptide nucleic acids designed for post-assembly conjugation reactions

Two orthogonally protected PNA monomers were prepared having the mercaptomethyl moiety attached to the PNA backbone. These building blocks were employed in solid-phase PNA synthesis and it was shown that Boc/S-p-methoxybenzyl protection scheme was only satisfactory for the introduction of N-terminal thiol modification while the Fmoc/S-butylthio protected monomer proved to be amenable to elongation. The mercaptomethyl modification did not influence the thermal stability of a PNA/RNA duplex. The feasibility of PNA-PNA native ligation was demonstrated.     

Paramagnetic Particles and PNA Probe for Automated Separation and Electrochemical Detection of Influenza

Considerable efforts have been devoted to the development of rapid and sensitive methods allowing the detection of viral nucleic acid. We herein describe an assay for identification of a specific influenza sequence. The suggested method was based on isolation using paramagnetic particles coupled with electrochemical detection of isolated product. Peptide nucleic acid (PNA) was used as a probe for hybridization and identification of the influenza-derived specific sequence. The use of PNA can show numerous benefits: PNA probe is not degradable by enzymes and the duplex of PNA with RNA/DNA is more thermostable and more resistant to pH changes than DNA/DNA or RNA/RNA duplexes. This PNA probe assay can be applied as a magnetically guidable tool for detection of DNA/RNA samples under different conditions.     

Optimization of the phase system in the analysis of polynuclear aromatics (PNA) from diesel engine exhaust by high performance liquid chromatography (HPLC)

Summary Polynuclear aromatics (PNA) were collected and enriched from diesel engine exhausts by means of a probe and an absorber system. Group separation of PNA from other constituents of exhaust was accomplished through elution with n-heptane on a silica column. A variety of phase systems ranging from unpolar to strong polar packings and eluents, respectively, were tested to resolve PNA into single species. Of these a n-octadecyl bonded silica packing and methanol/water was found to be the most optimal with regard to resolution for real PNA mixtures. Separation was completed within 2–3 h at isocratic conditions.Paper presented at Euroanalysis III, Dublin. August 20–25, 1978.     

Synthesis and characterization of (R)-miniPEG-containing chiral γ-peptide nucleic acids using the Fmoc strategy

Diethylene glycol (miniPEG)-containing chiral γPNA is considered to be one of the best PNA derivatives. Its preparation is mainly based on the Boc strategy for solid phase peptide synthesis (SPPS), requiring the repeated use of trifluoroacetic acid TFA, which is not suitable for the in situ synthesis of PNA arrays and some other applications under mild conditions. Herein, Fmoc/Cbz orthogonal protected miniPEG-containing chiral γPNA monomers were synthesized, and a 15mer γPNA was prepared using the Fmoc strategy under mild conditions.     

In situ, on-resin synthesis of 8-Br/NH2 adeninyl peptide nucleic acid (PNA) oligomers and complementation studies with DNA

A new, novel and efficient in situ synthesis of 8-aminoadeninyl PNA oligomers from corresponding 8-bromoadeninyl PNA oligomers is reported. The study of hybridisation properties of (8-Br/8-NH₂) PNA oligomers with cDNA reveals substitution-site dependent stabilization of derived triplexes and duplexes.     

(2S,5R/2R,5S)-Aminoethylpipecolyl aepip-aegPNA chimera: synthesis and duplex/triplex stability

This article reports the design and facile synthesis of novel chiral six-membered PNA analogues (2S,5R/2R,5S)-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic acid, aepipPNA IV that upon incorporation into standard aegPNA sequences effected stabilization of complexes with complementary target DNA. Substitution of aegPNA unit by the designed monomer at the C-terminus was more effective than substitution at N-terminus. The stabilizing behaviour improved with degree of substitution and was found to be dependent on their relative positions in the sequence. The six-membered piperidine ring in the design may freeze the rigid chair conformations and the relative stereochemistry of the substituents may in effect direct the complex formation with DNA/RNA by sequence-specific nucleobase recognition. In the present aepipPNA analogues, the l-trans stereochemical disposition of the substituents seems to lead to the favorable pre-organization of the PNA oligomers for complex formation with DNA. The results reported here further expand the repertoire of cyclic PNA analogues.     

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.     

Preliminary evaluation of electrochemical PNA array for detection of single base mismatch mutations

We report here a preliminary evaluation of a microfabricated disposable-type peptide nucleic acid (PNA) array, with a 20-channel electrode, for the detection of cancer gene c-Ki-ras. Synthetic 15-mer PNA probes complimentary to ras sequence modified with cysteine were immobilized on the gold electrodes on the array. The electrochemical PNA array was reacted with 20-mer oligonucleotide target or 128 bp PCR product for 1 h. The anodic current derived from an electrochemically active DNA binder Hoechst 33258 was measured using the PNA array in the 50 microL of reaction chamber. The anodic current from Hoechst 33258 increased with increasing the concentration of PCR product of ras gene in the range from 10(11) to 10(15) copy mL(-1). The single base mismatch mutations of c-Ki-ras/61 were also detected using the electrochemical PNA array.     

Synthesis and evaluation of (1S,2R/1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the torsion angle beta in the ethylenediamine segment to 60-70 degrees that is prevalent in PNA(2):DNA and PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer units was observed as a result of hybridization of PNA sequences with complementary DNA sequences. Hybridization studies with complementary DNA and RNA sequences using UV-T(m) measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S) isomer.     

New Synthetic Route to γ-Mercaptomethyl PNA Monomers

Peptide nucleic acids (PNAs) are oligonucleotide mimics widely used as antisense, antigene molecules, and biotechnological tools. Recently, several microarrays and other biosensors based on PNAs have been developed. The construction of PNA molecular beacons or light-up probes for DNA detection requires the labelling of the PNA moiety. Labels are usually attached at the C or N terminal end by a flexible linker or in the middle of a PNA sequence, substituting one PNA base with an artificial base or by attaching fluorophores to a modified PNA backbone. The need to develop simple protocols to label PNAs encouraged us to design a new procedure for the synthesis of γ-mercaptomethyl-modified PNA. Here we propose a new strategy for the synthesis of modified PNAs, bearing amino acid side chains. The synthesis is straightforward and is an improvement to the procedures reported so far, as it uses stable intermediates and proceeds with better yields.     

Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis

Peptide nucleic acid (PNA) oligomers can be used as probes in pre-gel hybridization experiments, as an alternative to Southern hybridization. In this technique, the PNA probe is hybridized to a cyanine-5 labeled DNA sample denatured at low ionic strength, and the mixture is directly injected for size separation into a capillary electrophoresis (CE) system equipped with laser-induced fluorescence (LIF) detector. The neutral backbone of PNA allows hybridization to occur at low ionic strength and assures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. We have used as a model system the cystic fibrosis R553X and R1162X single-base mutations and we have assessed the influence of various factors, such as temperature and denaturants concentration on DNA/PNA hybrid stability in order to achieve the high specificity required for a single base pair discrimination.     

Quantum chemical studies of peptide nucleic acid monomers and role of cyclohexyl modification on backbone flexibility

Peptide nucleic acids (PNA) bind sequence specifically to DNA/RNA and are of major interest for all fields of molecular biology and could form the basis for gene‐targeted drugs. Modifications are introduced in PNA to overcome problems associated with orientational selectivity in binding, to restrict conformational flexibility of backbone, and to discriminate binding for either DNA or RNA. The addition of geometrical isomers (1R,2S and 1S,2R) of cyclohexyl ring in the backbone of PNA could bring rigidification to PNA backbone and may impart specificity toward RNA. Therefore, quantum chemical studies are aimed to explore the conformational space, to find out preferred stable conformations of PNA and modified (1R,2S and 1S,2R) cyclohexyl PNA monomer. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009