Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs). High-performance ion-exchange chromatographic analysis

Peptide nucleic acids (PNAs) are oligonucleotide mimics containing a pseudopeptide chain, which are able to bind complementary DNA tracts with high affinity and selectivity. Two mixed-sequence PNA undecamers (1 and 2) were synthesized and their double-stranded adducts with the complementary oligonucleotides (3 and 4) were revealed by the appearance of the corresponding peak in anion-exchange HPLC. A DEAE column was used and elution was performed with aqueous Tris buffer (pH 8) and an ionic strength gradient (0-0.5 M NaCl). The same effect was not observed with non-complementary oligonucleotides. The stability of the PNA-DNA adducts under the conditions used in the chromatographic system was studied as a function of temperature. Furthermore, in competition experiments double-stranded oligonucleotides were challenged by a PNA complementary to one strand: the formation of the PNA-DNA hybrid and the displacement of the non-complementary strand were observed with high specificity. The results suggest a possible use of ion-exchange HPLC for studying PNA-DNA interactions, and indicate the efficiency of PNA probes in the chromatographic analysis of DNA.     

Influence of the Type of Junction in DNA-3′-Peptide Nucleic Acid (PNA) Chimeras on Their Binding Affinity to DNA and RNA

The automated on-line synthesis of DNA-3′-PNA (PNA=Polyamide Nucleic Acids) chimeras 1 – 3 is described, in which the 3′-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA either via a N-(2-mercaptoethyl)- (X=S), a N-(2-hydroxyethyl)- (X=O), or a N-(2-aminoethyl)- (X=NH) N-[(thymin-1-yl)acetyl]glycine unit. Furthermore, the DNA-3′-PNA chimera 4 without a nucleobase at the linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on duplex stabilization was determined to be in the order O>S≈NH, rendering the phosphodiester bridge the most favored linkage at the DNA/PNA junction. The observed strong duplex-destabilizing effects, when base mismatches or non-basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest that the base at the linking unit contributes significantly to duplex stabilization.     

Indigo Carmine as New Label in PNA Biosensor for Detection of Short Sequence of p53 Tumor Suppressor Gene

A new electrochemical PNA hybridization biosensor for detection of a 15‐mer sequence unique to p53 using indigo carmine (IC) as an electrochemical detector is described in this work. This genosensor is based on the hybridization of target oligonucleotide with its complementary probe immobilized on the gold electrode by self‐assembled monolayer formation. Because this label is electroactive in acidic medium, the interaction between IC and short sequence of p53 is studied by differential pulse voltammety (DPV) in 0.1 M H₂SO₄. The results of electrochemical impedance spectroscopy and cyclic voltammetry in the solution of [Fe(CN)₆]^3?/4? shows no breakage in PNA‐DNA duplex. A decrease in the voltammetric peak currents of IC is observed upon hybridization of the probe with the target DNA. The influence of probe concentration on effective discrimination against non‐complementary oligonucleotides is investigated and a concentration of 10^?7 M is selected. The diagnostic performance of the PNA sensor is described and the detection limit is found to be 4.31×10^?12 M.     

Hybridization energies of double strands composed of DNA, RNA, PNA and LNA

The electronic properties of double strands composed of trimeric LNA, PNA, DNA and RNA single strands were investigated by density-functional molecular orbital calculations. The computed hybridization energies for the double strands involving PNA or LNA are larger than those for DNA-DNA and RNA-RNA. The larger stability is attributed to the presence of a larger positive charge of the hydrogen atoms contributing to the hydrogen bonds in the PNA-DNA and LNA-DNA double-strands. These results are comparable to the experimental finding that PNA and LNA single strands display high affinity toward a complementary DNA or RNA single strand.     

(2′‐O‐Methyl‐RNA)‐3′‐PNA Chimeras: A New Class of Mixed Backbone Oligonucleotide Analogues with High Binding Affinity to RNA

The automated on‐line synthesis of DNA‐3′‐PNA chimeras 1 – 4 and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras 5 – 8 is described, in which the 3′‐terminal part of the oligonucleotide is linked to the N‐terminal part of the PNA via N‐(ω‐hydroxyalkyl)‐N‐[(thymin‐1‐yl)acetyl]glycine units (alkyl=Et, Ph, Bu, and pentyl). By means of UV thermal denaturation, the binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA and RNA. All investigated DNA‐3′‐PNA chimeras and (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras form more‐stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. Interestingly, a N‐(3‐hydroxypropyl)glycine linker resulted in the highest binding affinity for DNA‐3′‐PNA chimeras, whereas the (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras showed optimal binding with the homologous N‐(4‐hydroxybutyl)glycine linker. The duplexes of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras and RNA were significantly more stable than those containing the corresponding DNA‐3′‐PNA chimeras. Surprisingly, we found that the charged (2′‐O‐methyl‐RNA)‐3′‐PNA chimera with a N‐(4‐hydroxybutyl)glycine‐based unit at the junction to the PNA part shows the same binding affinity to RNA as uncharged PNA. Potential applications of (2′‐O‐methyl‐RNA)‐3′‐PNA chimeras include their use as antisense agents acting by a RNase‐independent mechanism of action, a prerequisite for antisense‐oligonucleotide‐mediated correction of aberrant splicing of pre‐mRNA.     

DNA Hydrolysis and Voltammetric Determination of Guanine and Adenine Using Different Electrodes

Abstract

This work reports the development of a biosensor method for the label‐free detection of specific DNA sequences. In the initial phase, square wave voltammetry (SWV) was used in a comparative investigation into the electrochemical oxidation of purines (guanine and adenine) and DNA fragments at various electrode surfaces: carbon paste (CPE), glassy carbon electrode (GCE), and gold (AuE). Relative to the carbon electrodes, an approximate 4.0‐fold, 6.0‐fold, and 3.25‐fold increase in the anodic response was observed when guanine, adenine, and hydrolyzed DNA, respectively, were measured on the AuE. It was shown that the guanine and adenine bases could be successfully determined by use of SWV for a deoxyribonucleic acid sample following acid hydrolysis. This label‐free detection of hydrolyzed DNA on gold electrodes has significant advantages over methods using existing carbon electrode materials because of its higher sensitivity and the potential applicability of microfabrication techniques for the production of the requisite gold electrodes.

In another phase of development, the times and conditions for DNA hydrolysis and purine release were investigated. It was shown that under optimal conditions, trace levels of the purine bases could be readily detected following 20 min of hydrolysis at room temperature. The proposed method can be used to estimate the guanine and adenine contents in DNA with in a linear range of 5–30 ng ml^?1.

Finally, when appropriate probe sequences were first adsorbed on the surface of the screen‐printed gold electrode (SPGE), this electrochemical biosensor could be used to specifically detect sequences from ss corona virus aviair following hybridization and hydrolysis reactions on the sensor surface. No enhancement of the voltammetric response was observed when the sensor was challenged with a non‐complementary DNA sequence.     

Detection of DNA immobilized on bare gold electrodes and gold nanoparticle-modified electrodes via electrogenerated chemiluminescence using a ruthenium complex as a tag

Electrogenerated chemiluminescence (ECL) for DNA hybridization detection is demonstrated based on DNA that was self-assembled onto a bare gold electrode and onto a gold nanoparticles modified gold electrode. A ruthenium complex served as an ECL tag. Gold nanoparticles were self-assembled on a gold electrode associated with a 1,6-hexanedithiol monolayer. The surface density of single stranded DNA (ssDNA) on the gold nanoparticle modified gold electrode was 4.8?×?10¹⁴ molecules per square centimeter which was 12-fold higher than that on the bare gold electrode. Hybridization was induced by exposure of the target ssDNA gold electrode to the solution of ECL probe consisting of complementary ssDNA tagged with ruthenium complex. The detection limit of target ssDNA on a gold nanoparticle modified gold electrode (6.7?×?10^?12 mol L^?1) is much lower than that on a bare gold electrode (1.2?×?10^?10 mol L^?1). The method has been applied to the detection of the DNA sequence related to cystic fibrosis. This work demonstrates that employment of gold nanoparticles self-assembled on a gold electrode is a promising strategy for the enhancement of the sensitivity of ECL detection of DNA.     

Thermodynamics and kinetics of PNA-DNA quadruplex-forming chimeras

PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to complementary single-strand (DNA or RNA), and the resistance to nuclease and protease degradation. At the same time, the limitations of an oligomer containing all PNA residues, such as low water solubility, self-aggregation, and low cellular uptake, are effectively overcome. Further, PNA-DNA chimeras possess interesting biological properties as antisense agents. We have explored the ability of PNA-DNA chimeric strands to assemble in quadruplex structures. The rate constant for association of the quadruplexes and their thermodynamic properties have been determined by CD spectroscopy and differential scanning calorimetry (DSC). Thermal denaturation experiments indicated higher thermal and thermodynamic stabilities for chimeric quadruplexes in comparison with the corresponding unmodified DNA quadruplex. Singular value decomposition analysis (SVD) suggests the presence of kinetically stable intermediate species in the quadruplex formation process. The experimental results have been discussed on the basis of molecular dynamic simulations. The ability of PNA-DNA chimeras to form stable quadruplex structures expands their potential utility as therapeutic agents.     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

Solid-State Polymerization in Binary Component Systems at Low Temperature

Abstract

Radiation-induced polymerization in binary component systems of acrylonitrile-methacrylonitrile and acrylonitrile-vinyl acetate was studied at ?196°C. A mixture of two-component homopolymers was obtained from the acrylonitrile-methacrylonitrile system, which forms a eutectic mixture. When the mixture of acrylonitrile with vinyl acetate is cooled quickly from room temperature, a glassy state can be obtained. It was found that the copolymerization is possible in the glassy state at ?196°C, and the monomer reactivity ratios were determined as r ₁ = 6.0 and r ₂ = 0.2 (M ₁ = acrylonitrile), which coincides with the reported values on the radical copolymerization at room temperature.     

Peptide Nucleic Acid Immobilized Biocompatible Silane Nanocomposite Platform for Mycobacterium tuberculosis Detection

We have prepared nanocomposite films comprising of 3‐glycidoxypropyltrimethoxysilane (GOPS) and iron‐oxide (Fe₃O₄) onto indium‐tin‐oxide (ITO) glass plate for covalent immobilization of 21‐mer peptide nucleic acid (PNA). These films have been characterized using contact angle, atomic force microscopy (AFM), electrochemical techniques. The electrochemical response of the GOPS/ITO and Fe₃O₄‐GOPS/ITO electrodes has been investigated by hybridization with complementary, non‐complementary and one‐base mismatch using methylene blue as electrochemical indicator. The PNA/Fe₃O₄‐GOPS/ITO bioelectrode exhibits improved specificity and detection limit (0.1 fM) as compared to that of the PNA‐GOPS/ITO bioelectrode (0.1 pM). This PNA/Fe₃O₄‐GOPS/ITO electrode can be utilized for detection of hybridization with the complementary sequence in sonicated M. tuberculosis genomic DNA within 90 s of hybridization time.     

Phosphotungstic salt used as an efficient catalyst to rapidly remove RhB from water solution

Ag-modified polyoxometalate [Na(H₂O)]₂[Ag(bpf)]₃[PW₁₂O₄₀] (1) (bpf = 2,4-di(4-pyridyl)furan) was synthesized through a hydrothermal reaction. Single-crystal X-ray diffraction analysis revealed that 1 consists of a hybrid 3D MOF constructed from Keggin-type polyanionic [PW₁₂O₄₀]^3- and cationic Ag-bpf units. Ligand bpf in 1 was in situ transferred from 1,3-bis(4-pyridyl)propane under the higher temperature and pressure of hydrothermal conditions. Catalytic performance of 1 as a heterogeneous catalyst for degradation of organic dye Rhodamine B (RhB) was investigated. Experimental results showed that hybrid 1 was especially active to catalytically degrade RhB under room temperature and natural light. RhB-containing solution (5.0 mg·L^?1) could be quickly bleached in a short time, and the high removal efficiency (97%) could be reached in a mere 30 min. The degradation mechanism of RhB was discussed.     

Nucleic acid sensor for M. tuberculosis detection based on surface plasmon resonance

Cysteine modified NH(2)-end peptide nucleic acid (PNA) (24-mer) probe and 5'-thiol end labeled deoxyribonucleic acid (DNA) probes specific to Mycobacterium tuberculosis have been immobilized onto BK-7 gold coated glass plates for the detection of complementary, one-base mismatch, non-complementary targets and complementary target sequence in genomic DNA of Mycobacterium tuberculosis using a surface plasmon resonance (SPR) technique. The DNA/Au and PNA/Au bio-electrodes have been characterized using contact angle, atomic force microscopy (AFM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetric (CV) techniques, respectively. It is revealed that there is a 252 millidegrees SPR angle change in the case of PNA immobilization and 205 millidegrees for DNA immobilization, indicating increased amount of immobilized PNA molecules. Hybridization studies reveal that there is no binding of the non-complementary target to DNA/Au and PNA/Au electrode. Compared to the DNA/Au bioelectrode, PNA/Au electrode has been found to be more efficient for detection of one-base mismatch sequence. The PNA/Au bioelectrode shows better detection limit (1.0 ng ml(-1)) over the DNA-Au bioelectrode (3.0 ng ml(-1)). The values of the association (k(a)) and dissociation rate constant (k(d)) for the complementary sequence in case of the PNA/Au bioelectrode have been estimated as 8.5 x 10(4) m(-1) s(-1) and 3.6 x 10(-3) s(-1), respectively.     

Ionic liquids from the cationic cobalt(III) Schiff base complex, [Co(acacen)L2][Tf2N] (acacen = N,N′-bis(acetylacetone)ethylenediamine,Tf2N = bis(trifluoromethanesulfonyl)amide)

Ionic liquids comprising cationic cobalt(III) complexes [Co(acacen)L₂][Tf₂N] (L?=?3-butylpyridine (1), 1-butylimidazole (2); acacen?=?N,N′-bis(acetylacetone)ethylenediamine, Tf₂N?=?bis(trifluoromethanesulfonyl)amide) were prepared. 1 is a liquid at room temperature and exhibits a glass transition at ?12?°C, whereas 2 is a solid at room temperature with a melting point of 74.6?°C and glass transition temperature of ?15?°C upon cooling from the melt. These salts are reddish brown diamagnetic materials that are stable against air and water; these properties differ from those of the corresponding iron(III) salt. Desorption of the axial ligands of 1 and 2 occurs at 180 and 207?°C, respectively.     

Colloidal Gold Nanoparticles Protected by Cationic Polyelectrolytes

Abstract

Several stable gold colloids were prepared by the in-situ reduction of hydrogen tetrachloroaurate (HAuCl₄) in the presence of various cationic polyelectrolytes. Several types of such polyelectrolytes were investigated for their ability to stabilize gold colloids, and UV – VIS spectroscopy was used to follow the in-situ reductions and to further characterize the colloids. The particle sizes and size distributions were determined by transmission electron microscopy (TEM). TEM micrographs and UV-VIS spectra were also used to characterize the stability of the colloids after storage for nine months in air at room temperature. Colloids protected by the cationic polyelectrolytes with ammonium side-groups along a hydrophobic polymer backbone frequently exhibited very good stability.     

A porous Cu(II)-MOF containing [PW12O40]3− and a large protonated water cluster: synthesis,structure, and proton conductivity

A proton-conducting metal–organic framework (MOF), {[Cu₄(dpdo)₁₂][H(H₂O)₂₇(CH₃CN)₁₂][PW₁₂O₄₀]₃}_n (where dpdo is 4,4′-bipyridine-N,N′-dioxide) (1), was synthesized by the reaction of CuHPW₁₂O₄₀·nH₂O and dpdo at room temperature. Single-crystal X-ray diffraction analysis at 293?K revealed that 1 crystallized in the cubic space group Im-3 and presented a non-interwoven 3-D framework with cubic cavities and guest molecules. A large ionic water cluster H⁺(H₂O)₂₇, consisting of a water shell (H₂O)₂₆ and an encaged H⁺(H₂O) as a center core, was trapped in the cubic cavity of the MOF {[Cu₄(dpdo)₁₂(PW₁₂O₄₀)₃]^?}_∞. Thermogravimetric analysis suggests that 1 has high thermal stability, indicating that such a non-interwoven 3-D framework with cubic cavities is a suitable host for researching protonated water clusters. Its water vapor adsorption isotherm at room temperature and pressure shows that the water vapor adsorbed in it was 65.1 cm^3?g^?1 at the maximum allowable humidity. It exhibits good proton conductivities of 10^?5–10^?4?S^?cm^?1 at 100 °C in the relative humidity range 35–98%.     

Hybridization of pyrrolidinyl peptide nucleic acids and DNA: selectivity, base-pairing specificity, and direction of binding

[structure: see text] A mixed-base, beta-amino acid containing, pyrrolidinyl peptide nucleic acid (PNA) binds strongly and selectively to complementary DNA in an exclusively antiparallel fashion. The PNA-DNA binding specificity strictly follows the Watson-Crick base-pairing rules.     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

Silica‐Supported Sodium Hydrogen Sulfate Catalyzed Facile Transformation of p‐Hydroxybenzyl Alcohols to p‐Hydroxybenzyl Ethers and Thioethers

Abstract

The heterogeneous catalyst, silica‐supported sodium hydrogen sulfate (NaHSO₄ · SiO₂) has been found to be highly efficient in carrying out the transformation of p‐hydroxybenzyl alcohols at room temperature to p‐hydroxybenzyl ethers and thioethers in very high yields.     

Molecular-beacon-based tricomponent probe for SNP analysis in folded nucleic acids

Hybridization probes are often inefficient in the analysis of single‐stranded DNA or RNA that are folded in stable secondary structures. A molecular beacon (MB) probe is a short DNA hairpin with a fluorophore and a quencher attached to opposite sides of the oligonucleotide. The probe is widely used in real‐time analysis of specific DNA and RNA sequences. This study demonstrates how a conventional MB probe can be used for the analysis of nucleic acids that form very stable (T_m>80 °C) hairpin structures. Here we demonstrate that the MB probe is not efficient in direct analysis of secondary structure‐folded analytes, whereas a MB‐based tricomponent probe is suitable for these purposes. The tricomponent probe takes advantage of two oligonucleotide adaptor strands f and m. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to a MB probe. In the presence of a specific analyte, the two adaptor strands hybridize to the analyte and the MB probe, thus forming a quadripartite complex. DNA strand f binds to the analyte with high affinity and unwinds its secondary structure. Strand m forms a stable complex only with the fully complementary analyte. The MB probe fluorescently reports the formation of the quadripartite associate. It was demonstrated that the DNA analytes folded in hairpin structures with stems containing 5, 6, 7, 8, 9, 11, or 13 base pairs can be detected in real time with the limit of detection (LOD) lying in the nanomolar range. The stability of the stem region in the DNA analyte did not affect the LOD. Analytes containing single base substitutions in the stem or in the loop positions were discriminated from the fully complementary DNA at room temperature. The tricomponent probe promises to simplify nucleic acid analysis at ambient temperatures in such applications as in vivo RNA monitoring, detection of pathogens, and single nucleotide polymorphism (SNP) genotyping by DNA microarrays.