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 photoactivity of phenylazonaphthalene peptide nucleic acid monomers

Phenylazonaphthalene peptide nucleic acid （PNA） monomers were successfully synthesized, and their photoisomerization was examined. The new PNA monomers showed reversible trans-cis isomerization with UVand visible light irradiation, which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA. Simultaneously, the fluorescence of the new PNA monomers might make them especially useful as structural probes.     

Design of a new oligotriazole peptide nucleic acid analogue (oT-PNA)

We describe in this Letter the synthesis of an original thymine azido-heterotrimer generated by Click Chemistry. This trimer has been obtained from an azido-thymidine and a new chloroethyl-propargylated PNA monomer analogue, after two azidation/click-reaction cycles. Conformational preferences of a rotameric intermediate have also been studied     

High sensitivity detection of 16s rRNA using peptide nucleic acid probes and a surface plasmon resonance biosensor

A signal enhancing method allowing highly sensitive detection of E. coli 16s rRNA was developed using peptide nucleic acid (PNA) as a capture probe and a surface plasmon resonance (SPR) sensor as a detector. 16s rRNA has been used as a genetic marker for identification of organisms, and can be analyzed directly without PCR amplification due to the relatively high number of copies. PNA has a neutral backbone structure, therefore hybridization with 16s rRNA results in the ionic condition being changed from neutral to negative. A cationic Au nanoparticle was synthesized and used for signal amplification by ionic interaction with 16s rRNA hybridized on the PNA probe-immobilized SPR sensor chip. This method resulted in a detection limit of E. coli rRNA of 58.2 ± 1.37 pg mL⁻¹. Using this analytical method, Staphylococcus aureus was detected without purification of rRNA.     

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.     

Synthesis of luminescent 2,3-diphenylmaleimide-labelled peptide nucleic acid oligomers

A lys-GTAGATCACT-lys peptide nucleic acid (PNA) decamer labelled with the luminescent 2,3-diphenyl maleimido (DPM) group on the ε-position of the terminal lysine residue was prepared through an automated solid phase synthesis. Fluorescence emission of the DPM-labelled PNA thus obtained was found to be significant and promising for the potential application in DNA recognition.     

New synthesis of a spin-labeled peptide nucleic acid and its interactions with nucleic acids

A new combined solid-liquid phase synthesis method for a spin labeled peptide nucleic acid (PNA) is developed. The methodology involved initial preparation of a protected PNA on solid phase, followed by efficient solution phase coupling to a spin label containing a reactive carboxylic group. This strategy allows to maintain the integrity of the nitroxide moiety during the various steps of chemical synthesis assuring in the same time the fidelity of the hybridization assay. This compound can be used as a reporter molecule to investigate the binding of peptide nucleic acids to oligonucleotide sequences (DNA or RNA) by EPR spectroscopy.     

Synthesis and polymerisation of lipophilic peptide nucleic acids derived from stearic acid and pentacosa-10,12-diynoic acid

The adenine, cytosine and thymine peptide nucleic acid (PNA) monomers and PNA T₁₀ oligomers bearing either a diacetylenic or stearoyl moiety at the N- or C-terminus have been successfully prepared. The resulting thymine monomeric and T₁₀-mer derivatives have been subsequently incorporated into polydiacetylene-containing liposomes.     

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs)

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5–10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R² = 0.990, which was comparable to the value of R² = 0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.     

Versatile biosensor surface based on peptide nucleic acid with label free and total internal reflection fluorescence detection for quantification of endocrine disruptors

In recent years, the simultaneous detection of hundreds of substances has become increasingly important in medical diagnostics and environmental monitoring. This calls for methods that allow fast and simultaneous measurement of many analytes and require only a minimum of attendance. Biosensor-applications including easy implementation and long-term stability of the sensor element are more and more designed to meet these demands.In this paper, a surface based on covalently immobilised peptide nucleic acid (PNA) for the detection of different endocrine disruptors is used. The surface was characterised with a label free detection system, the reflectometric interference spectroscopy (RIfS). A hybridisation capacity with DNA oligonucleotides of 1.3 ng/mm² (180 fmol/mm²) on PNA-surfaces was achieved. The PNA transducer is stable for half a year and for more than 300 regeneration steps.With this modified surface various environmentally relevant endocrine disruptors could be detected by an immunoassay. The detection is done on an optical waveguide system, based on total internal reflection fluorescence (TIRF) by using an auxiliary-system consisting of a conjugate which is formed of DNA-oligomers and analyte-derivatives. The successive detection of different analytes on the same spot by using this auxiliary-system is demonstrated as one application of multianalyte detection. Quantification of different endocrine disruptors on the same PNA-surface is demonstrated by three calibration curves.     

Sensitive detection of a serum biomarker based on peptide nucleic acid-coupled dual cycling reactions

Serum level of disease markers may provide important guidance for diagnosis and prognosis. In this work, a sensitive and specific method suitable for direct serum detection of biomarkers is developed based on peptide nucleic acid (PNA)-coupled DNA cycling reactions with dual amplification. In this method, PNA released from a target-triggered homogeneous DNA cycling is employed to initiate an interface DNA cycling, and both of the cycling reactions are based on polymerase-assisted strand displacement reaction. Consequently, two PNA-coupled DNA cycling steps can take place simultaneously in one-pot, leading to greatly enhanced limit of detection and simplified operation. This method has also been successfully applied for evaluating serum insulin in pregnant women as an indicator of gestational diabetes mellitus. So the application of this method in real bio-samples may allow it to hold considerable potential in clinical practice. In addition, since there is no requirement for specific sequence of aptamer, the strategy proposed can be extended for the detection of many other protein markers and peptide-hormones in the future.     

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.     

Synthesis of N-propynyl analogues of peptide nucleic acid (PNA) monomers and their use in the click reaction to prepare N-functionalized PNAs

Application of the click reaction for coupling a 2-(2-aminoethoxy)ethoxy (AEE) function to thyminyl, cytosinyl and adeninyl peptide nucleic acid (PNA) monomer analogues bearing a N-propynyl group, in place of the original N-2-aminoethyl moiety, has been explored. The N-propynyl PNA analogues were prepared from glycine ethyl ester hydrochloride and the structure of the thyminyl derivative was confirmed by X-ray diffraction. These monomers were employed in click reactions with Boc-protected AEE azide to afford the corresponding triazolyl-linked PNA-AEE conjugates in yields ranging from 64 to 76%. [1,4]-Regiochemistry was verified from a NOESY correlation experiment.     

核酸、蛋白质的电荷数随pH的变化规律

从理论上推导出核酸、蛋白质的正电荷数随pH的变化关系式:Y=∑(i=1-3)[H^+]~n~i/([H^+]+K~b~i),负电荷数随pH的变化关系式:X=∑(i=1-5)K~a~im~i/([H^+]+k~a~i)、净电荷数随pH变化关系式以及等电点计算关系式,并从关系式中导出核苷酸、碱基、中性氨基酸、酸性氨基酸、碱性氨基酸的等电点关系式,表明理论推导是正确、可靠的。用表面活性剂沉淀肌酸激酶、不同pH的溶菌酶电泳方法验证了计算结果的可靠性。同时,计算了人DNA、蚕DNA、β-乳球蛋白带电荷数随pH的变化规律,并估计了计算误差。     

On the relative acidity and basicity of the amino groups of the nucleic acid bases

Quantum chemical calculations are reported on the deprotonation and protonation of the amino groups of the nucleic acid bases adenine, guanine and cytosine, in an attempt to compare the relative reactivities of these groups. In the light of renewed interest in the amino groups as reactive sites for certain carcinogenic and carcinostatic agents, we discuss the possible significance of our results for the interpretation of these molecular interactions.     

Combining G-quadruplex targeting motifs on a single peptide nucleic acid scaffold: a hybrid (3+1) PNA-DNA bimolecular quadruplex

We describe the first G-quadruplex targeting approach that combines intercalation and hybridization strategies by investigating the interaction of a G-rich peptide nucleic acid (PNA) acridone conjugate 1 with a three-repeat fragment of the human telomere G 3 to form a hybrid PNA-DNA quadruplex that mimicks the biologically relevant (3+1) pure DNA dimeric telomeric quadruplex. Using a combination of UV and fluorescence spectroscopy, circular dichroism (CD), and mass-spectrometry, we show that PNA 1 can induce the formation of a bimolecular hybrid quadruplex even at low salt concentration upon interaction with a single-stranded three-repeat fragment of telomeric DNA. However, PNA 1 cannot invade a short fragment of B-DNA even if the latter contains a CCC motif complementary to the PNA sequence. These studies could open up new possibilities for the design of a novel generation of quadruplex ligands that target not only the external features of the quadruplex but also its central core constituted by the tetrads themselves.     

Force field validation for nucleic acid simulations: comparing energies and dynamics of a DNA dodecamer

Important questions exist regarding the quality of force fields used in molecular dynamics (MD) simulations and their interoperable use with other available MD implementations. NAMD is one of the most efficient and scalable parallel molecular dynamics codes for large-scale biomolecular simulations in the open source domain. It is the aim of this article to analyze and compare the dynamics of a benchmark DNA dodecamer d(CTTTTGCAAAAG)2 system, including its binding to a specific drug molecule arising from the use of various simulation protocols in NAMD using Amber98, with the dynamics arising from simulations of the same dodecamer using Amber98 in the AMBER package, one of the most well-established simulation codes for nucleic acids. Based upon a set of validation benchmarks, the details of which are discussed, we find that nucleic acid simulations using NAMD give meaningful results and that the essential features of the resulting dynamics are similar to those arising from the AMBER package. This sets the stage for reliable large-scale simulations of nucleic acids using NAMD.     

Synthesis of pyrrolidine-based oxy-peptide nucleic acids carrying four types of nucleobases and their transport into cytoplasm

We synthesized 16 pyrrolidine-based oxy-peptide nucleic acid (POPNA) monomers carrying four different nucleobases onto four different stereoisomers of pyrrolidine rings. Using these monomers, we prepared POPNA oligomers, which formed sequence-specific hybrids with DNAs. The oligomer configurations influenced the hybrid stability. The oligomers were not taken into CHO cells. However, they could enter the cell cytoplasm when mixed with the influenza virus hemagglutinin peptide-arginine heptamer conjugate.