Stabilisation of nucleic acid secondary structures by oligonucleotides with an additional nucleobase; synthesis and incorporation of 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine

A nucleoside with two nucleobases is incorporated into oligonucleotides. The synthetic building block, 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine, 2, is prepared from uridine via 5',3'-TIPDS-protected 2'-deoxy-2'-C-allyluridine by an oxidative cleavage of the allyl group, a Mitsunobu reaction for the introduction of thymine and appropriate deprotection reactions. This compound is converted into a DMT-protected phosphoramidite and incorporated once into a 13-mer oligodeoxynucleotide sequence, once in an isosequential LNA-modified oligodeoxynucleotide and four times in the middle of a 12-mer oligodeoxynucleotide. These sequences are mixed with different complementary DNA and RNA sequences in order to study the effect of the additional nucleobase in duplexes, in bulged duplexes and in three-way junctions. The first additional thymine is found to be well-accommodated in a DNA-RNA duplex, whereas a DNA-DNA duplex was slightly destabilised. A three-way junction with the additional thymine in the branching point is found to be stabilised in both a DNA-DNA and a DNA-RNA context but destabilised where the modified LNA-sequence is used. In a Mg2+-containing buffer, however, the relative stability of the three-way junctions is found to be opposite with especially the LNA-modified DNA-DNA complex being significantly stabilised by the additional nucleobase.     

Fluorescence and hybridization properties of peptide nucleic acid containing a substituted phenylpyrrolocytosine designed to engage Guanine with an additional H-bond

A new pyrrolocytosine derivative has been designed to selectively interact with guanine and has been evaluated in peptide nucleic acid where it imparts increased selective binding affinity for complementary oligonucleotides. The modified nucleobase also possesses an exceptionally high fluorescence quantum yield that is responsive to hybridization.     

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.     

Photoelectrochemical properties of Fe2O3-Nb2O5 films prepared by sol-gel method

Fe2O3-Nb2O5 coating films of various Nb/(Fe + Nb) mole ratios were prepared on nesa silica glass substrates from Fe(NO3)3.9H2O - NbCl5 - CH3(CH2)2CH2OH - CH3COOH solutions by the sol-gel method. The photoanodic properties were studied in a three-electrode cell with an aqueous buffer solution of pH = 7 as the supporting electrolyte. The crystalline phases identified were alpha-Fe2O3 (Nb/(Fe + Nb) = 0), alpha-Fe2O3 + FeNbO4 (Nb/(Fe + Nb) = 0.25), FeNbO4 (Nb/(Fe + Nb) = 0.5), FeNbO4 + Nb2O5 (Nb/(Fe + Nb) = 0.75), and Nb2O5 (Nb/(Fe + Nb) = 1). When the Nb/(Fe + Nb) mole ratio increased from 0 to 0.25, the crystalline phases changed from alpha-Fe2O3 to alpha-Fe2O3 + FeNbO4, the photoanodic current under white light illumination increased, and the photoanodic current under monochromatized light illumination increased in both visible and ultraviolet regions. When the Nb/(Fe + Nb) ratio increased over 0.25, the crystalline phases changed to FeNbO4, FeNbO4 + Nb2O5, or Nb2O5, and the photoanodic current decreased. The sample consisting of alpha-Fe2O3 and FeNbO4 (Nb/(Fe + Nb) = 0.25) exhibited photoresponse extending to 600 nm and an IPCE of 18% at a wavelength of 325 nm.     

Synthesis and properties of a novel bridged nucleic acid with a P3'' --> N5'' phosphoramidate linkage, 5''-amino-2'',4''-BNA

5'-Amino-2',4'-BNA, a novel analogue of BNA series compounds, was successfully synthesized, and its incorporated oligonucleotides showed potent duplex- and triplex-forming ability and resistance against snake venom phosphodiesterase.     

Surface modification with phosphoric acid of SiO2/Nb2O5 prepared by the sol-gel method: structural-textural and acid sites studies and an ion exchange model

In this work, the structural and textural properties of the SiO2/Nb2O5 system prepared by the sol-gel method and then modified by phosphoric acid were studied. The different materials were prepared, with three different mol % Nb2O5 (2.5, 5.0, and 7.5 mol %), and calcined in the temperature range of 423-1273 K. BET specific surface area determinations, scanning electron microscopy connected to a X-ray emission analyzer, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy (XPS) were used for the investigation. For the lowest temperature of calcination (423 K), the mesopores and micropores of the modified material were blocked, resulting in a decrease of the specific surface area compared to the SBET values obtained for the SiNb matrix. Under intermediate temperatures of calcination (423-873 K), the modified material acquired textural stability. By XPS analysis, the presence of the dihydrogenphosphate species was identified, the P/Nb atomic ratios being independent of the thermal treatment. 31P magic angle spinning NMR confirmed the XPS data and also showed that the chemical shift of the (H2PO4)- ions strongly depended on the crystallization degree of the Nb2O5. Structural thermal stability was also shown by the presence of Br?nsted acid sites in the modified material calcined at high temperature (1273 K). The thermal stability is directly associated with obtainment of the same value for K+ exchange capacity (0.74 mmol g(-1), average value) for the modified materials calcined at 423 and 1273 K. The chemical analyses of phosphorus for the modified materials were made by using the inductively coupled plasma. The value was 0.36 mmol g(-1), corroborating the presence of (H2PO4)- ions. The ion exchange isotherms presented an S-shaped form characteristic of energetically heterogeneous ion exchangers, permitting application of a model of fixed polydentate centers, in which ion exchange took place.     

Luminescent properties of pure cubic phase Y2O3/Eu3+ nanotubes/nanowires prepared by a hydrothermal method

One-dimensional pure cubic Y(2)O(3)/Eu(3+) nanocrystals (NCs) were synthesized by a hydrothermal method at various temperatures. The NCs prepared at 130 degrees C yielded nanotubes (NTs) with wall thickness of 5-10 nm and outer diameter of 20-40 nm. The NCs prepared at 170 and 180 degrees C yielded nanowires (NWs) with diameters of approximately 100 and approximately 300 nm, respectively. Their luminescent properties, including electronic transition processes, local environments surrounding Eu(3+) ions, electron-phonon coupling, and UV light irradiation induced spectral changes have been systematically studied and compared. The results indicate that the Y(2)O(3)/Eu(3+) NTs and NWs have strong red (5)D(0)-(7)F(2) transitions. The fluorescence lifetime of (5)D(1)-(7)F(1) hardly changes in different samples, while that of (5)D(0)-(7)F(2) decreases a small amount in Y(2)O(3)/Eu(3+) NTs. The (5)D(0)-(7)F(2) lines originate from the emissions of Eu(3+) ions occupying one C(2) site, like that in the bulk powders. The phonon sideline with a frequency shift of 40-50 cm(-1) appears at the low-energy side of the (7)F(0)-(5)D(0) zero phonon line. The relative intensity of the sideline to zero phonon line increases by varying from NTs to NWs, and the spectral position of the phonon sideline shifts red. The UV light irradiation induced spectral change in the charge-transfer band was studied. The results indicate that the spectral change is dependent on sample size and is wavelength selective. A detailed model was proposed to explain the light-induced spectral change.     

Surface-enhanced Raman spectrometry of triamterene on a silver substrate prepared by the nitric acid etching method

Surface-enhanced Raman scattering (SERS) spectrometry of the diuretic drug triamterene is discussed. The SERS-active substrate used is a silver foil etched with nitric acid. The influence of solvent and sample doping method on sensivity, intercept and shape of the calibration graphs is discussed.     

Determination of urinary 5-hydroxy-3-indoleacetic acid by an automated HPLC method

A HPLC method for the quantitative determination of 5-hydroxy-3-indoleacetic acid (5-HIAA) in urine is described. The method is based on ion-pair chromatography, reversed phase (RP) column material and specific fluorimetric detection at 300 nm and 355 nm. Sample preparation and gradient elution were avoided by using a column-switching technique. The sensitivity of the assay was excellent for clinical routine analysis, with a detection limit of 0.2 mg/L 5-HIAA. No endogenous or exogenous interference problems arose. Intra- and interassay precision was good, with observed coefficients of variation of 1.5 to 2.6% and 2.1%, respectively. Recoveries were 93 to 98%. The system described can be used for clinical diagnosis and therapy follow-up of carcinoid tumors. It has been running for over a year without disturbances and with a minimum of technical attendance.     

Rapid determination of orotic acid in urine by a fast liquid chromatography/tandem mass spectrometric method

Quantification of orotic acid (uracil-6-carboxylic acid) in urine is an important tool to diagnose some inherited diseases, such as urea cycle disorder (OTCD) and hereditary orotic aciduria. New rapid analytical methods are necessary to provide high-throughput orotic acid analyses. A new analytical method has been developed for the rapid analysis of orotic acid in urine by liquid chromatography coupled with ion spray tandem mass spectrometry (LC/MS/MS). After a sample dilution 1:20, the analysis was performed in the selected reaction monitoring mode in which orotic acid was detected through the transition m/z 155 to 111. The retention time was 3.9 min in a 4.5-min analysis. Daily calibration between 0.5-5.0 micromol/L of orotic acid, corresponding to 10-100 micromol/L in urine before the 1:20 dilution, offered consistent linearity and reproducibility. Interassay coefficient of variance (c.v.) was 4.97% at a mean concentration of 10.99 micromol/L. The sensitivity and specificity of tandem mass spectrometry permitted a high volume of analyses of orotic acid. The sample preparation is simple, inexpensive and not time demanding.     

Hydrotreating performance of La-modified Ni/Al2O3 catalysts prepared by hydrothermal impregnation method

Kinetics and Catalysis - To enhance the surface active sites of the catalyst, a series of La-modified Ni/γ-Al2O3 samples were prepared by hydrothermal impregnation method and characterized by...     

Characterization and properties of Eu3+-doped CdWO4 prepared by a hydrothermal method

Eu³⁺-doped CdWO₄ was prepared for the first time by a hydrothermal method. The structure, morphology, and luminescence of the Eu³⁺-doped CdWO₄ were characterized. TEM results revealed that the pure CdWO₄ was a nanorod with a width of about 50 nm. The photoluminescent properties of Eu³⁺-doped CdWO₄ complexes indicated energy transfer from WO₄ ^2? groups to Eu³⁺ and suggested effective doping of Eu³⁺ into the lattice of CdWO₄. The photocatalytic activity of CdWO₄ and Eu³⁺-doped CdWO₄ was investigated by the photodegradation of methyl orange (MO). Eu³⁺-doped CdWO₄ had enhanced photocatalytic activity in the photodegradation of MO. The hydroxyl radical was detected by the terephthalic acid photoluminescence (TA-PL) method, and the regular change revealed that the hydroxyl radical may be the active species.     

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.     

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

In order to recognize the presence of the R553X point mutation of the cystic fibrosis (CF) gene in the human genome, a peptide nucleic acid (PNA) complementary to the mutated gene tract and bearing three adjacent chiral monomers based on D-lysine (chiral box) was synthesized and used as a probe in CE. Binding specificity was preliminarily studied with complementary and mismatched oligonucleotides by UV spectroscopy, electrospray MS, and electrophoresis, indicating a very high sequence selectivity. The chiral PNA probe was then hybridized to cyanine-5-labeled DNA samples (186 bp), obtained by PCR amplification, respectively, from: (a) normal homozygous subjects (wtDNA), (b) CF-affected homozygous subjects (mutDNA), (c) heterozygous subjects (healthy carriers) and denatured at low ionic strength. The PNA-DNA mixture was directly analyzed by CE with LIF detection: a new signal corresponding to the PNA-mutDNA duplex was observed, in the case of CF-affected homozygous subjects, whereas for the sample containing the mismatched sequence (normal homozygous wtDNA) only the signal corresponding to ssDNA (ss, single strand) was detected. In the case of heterozygous DNA, both PNA-mutDNA duplex and ssDNA were detected. With this simple assay, it was possible to discriminate in an easy way among the three cases (mutated homozygous, normal homozygous, and heterozygous subjects) with a total specificity, thus allowing a decisive advance for the diagnosis of CF.     

The effect of formulation variables on the characteristics of insulin-loaded poly(lactic-co-glycolic acid) microspheres prepared by a single phase oil in oil solvent evaporation method

Biodegradable polymeric microspheres are ideal vehicles for controlled delivery applications of drugs, peptides and proteins. Amongst them, poly(lactic-co-glycolic acid) (PLGA) has generated enormous interest due to their favorable properties and also has been approved by FDA for drug delivery. Insulin-loaded PLGA microparticles were prepared by our developed single phase oil in oil (o/o) emulsion solvent evaporation technique. Insulin, a model protein, was successfully loaded into microparticles by changing experimental variables such as polymer molecular weight, polymer concentration, surfactant concentration and stirring speed in order to optimize process variables on drug encapsulation efficiency, release rates, size and size distribution. A 2⁴ full factorial design was employed to evaluate systematically the combined effect of variables on responses. Scanning electron microscope (SEM) confirmed spherical shapes, smooth surface morphology and microsphere structure without aggregation. FTIR and DSC results showed drug–polymer interaction. The encapsulation efficiency of insulin was mainly influenced by surfactant concentration. Moreover, polymer concentration and polymer molecular weight affected burst release of drug and size characteristics of microspheres, respectively. It was concluded that using PLGA with higher molecular weight, high surfactant and polymer concentrations led to a more appropriate encapsulation efficiency of insulin with low burst effect and desirable release pattern.     

Characterization of Zr-doped TiO2 nanocrystals prepared by a nonhydrolytic sol-gel method at high temperatures

Highly crystalline and surface-modified Zr-doped TiO(2) nanorods were successfully prepared using a nonhydrolytic sol-gel method that involves the condensation of metal halides with alkoxides in anhydrous trioctylphosphine oxide (TOPO) at either 320 or 400 degrees C. In addition, the interaction of the cross-condensation between the Ti and Zr species was studied by characterizing the morphologies, crystalline structures, chemical compositions, surface properties, and band gaps of the nanocrystals obtained at different reaction temperatures and Zr-to-Ti stoichiometric ratios. Increases in the concentration of Zr(4+) and in the reaction temperature led to large nanorods and regular shapes, respectively. In addition, only the anatase form was observed in the Zr-doped TiO(2) nanorods. The Zr-to-Ti ratios obtained ranged from 0.01 to 2.05, all of which were far below the stoichiometric ratios used during the preparation of the samples (0.25-4). Moreover, the Zr(4+) units accumulated mainly at the surface of the TiO(2) nanocrystals. The band gaps of the Zr-doped TiO(2) nanorods ranged from 2.8 to 3.8 eV, which are smaller than those of pure TiO(2) (3.7 eV) or ZrO(2) (5.2 eV). The Zr-doped anatase TiO(2) nanorods prepared at 400 degrees C at an initial stoichiometric Zr-to-Ti ratio of 2:3 exhibited the highest photoactivities for the decomposition of rhodamine B because of the presence of trace amounts of Zr(4+) (Zr/Ti = 0.03) in the TiO(2) and the regular shapes of these particles. DSC analysis indicated that the temperatures for forming nanocrystalline TiO(2) and ZrO(2) were 207 and 340 degrees C, respectively. Moreover, the reactivities of condensation between the Ti species were reduced when Zr species were involved in the NHSG reactions. The results obtained in this study clearly demonstrate that the faster kinetics for the generation of TiO(2) controls the material properties as well as the photoactivities of the nonhydrolytic sol-gel-derived nanocrystals.     

一步水热合成Y-Co3O4复合氧化物催化剂及催化分解N2O

用一步水热、分步水热、浸渍等方法分别制备Y-Co₃O₄复合氧化物,用于催化分解N₂O的反应,其中,一步水热法制备的催化剂活性较高。再用一步水热法制备了不同Y/Co物质的量比的Y-Co₃O₄复合氧化物,在优化出的催化剂（0.03Y-Co₃O₄）表面浸渍K₂CO₃溶液,制备K改性催化剂（0.02K/0.03Y-Co₃O₄）。用X射线衍射（XRD）、N₂物理吸附、H₂程序升温还原（H₂-TPR）、O₂程序升温脱附（O₂-TPD）、扫描电镜（SEM）、X射线光电子谱（XPS）等技术表征催化剂结构。研究发现,Co₃O₄和Y-Co₃O₄同为尖晶石结构,但Y-Co₃O₄的催化活性显著高于Co₃O₄。K改性增加了催化剂表面的活性位（Co²⁺）,还有利于吸附氧的脱除,从而提高了催化剂活性。在无氧无水、有氧无水、有氧有水气氛中,K改性催化剂上的N₂O全分解温度分别为325、350、375 ℃,催化剂活性较高。有氧有水气氛350 ℃连续反应50 h,K改性催化剂上N₂O分解率保持90%以上,稳定性较高。研究发现,Y-Co₃O₄及K改性催化剂上N₂O分解反应的E_a和lnA之间存在动力学补偿效应。