DNA-Binding and cytotoxicity of the cobalt(III) ethylenediamine pyrazole complex [Co(en)2(pyz)2]3+

The complex of cobalt(III) ethylenediamine was synthesized, isolated and characterized by UV-Vis, IR, and 1H NMR spectral methods. The binding of the complex with calf thymus DNA was investigated by absorption and emission spectroscopy, viscosity measurements, DNA melting and DNA photocleavage. The spectroscopic studies together with the viscosity measurements and DNA melting studies indicated that the complex binds to calf thymus DNA in a nonintercalative mode. This complex is found to promote photocleavage of the DNA plasmid pBR322 and shows a cytotoxic effect against CHO cells.     

Molecular dynamics studies of ion distributions for DNA duplexes and DNA clusters: salt effects and connection to DNA melting

We present extensive molecular dynamics simulations of the ion distributions for DNA duplexes and DNA clusters using the Amber force field with implicit water. The distribution of ions and the electrostatic energy of ions around an isolated DNA duplex and clusters of DNA duplexes in different salt (NaCl) concentrations over the range 0.2-1.0 mol/L are determined on the basis of the simulation results. Using the electrostatic energy profile, we determine a local net charge fraction phi, which is found to increase with increasing of salt concentration. For DNA clusters containing two DNA duplexes (DNA pair) or four DNA duplexes, phi increases as the distance between the duplexes decreases. Combining this result with experimental results for the dependence of the DNA melting temperature on bulk salt concentration, we conclude that for a pair of DNA duplexes the melting temperature increases by 5-10 K for interaxis separations of 25-40 A. For a cluster of four DNA duplexes, an even larger melting temperature increase should occur. We argue that this melting temperature increase in dense DNA clusters is responsible for the cooperative melting mechanism in DNA-linked nanoparticle aggregates and DNA-linked polymer aggregates.     

Sensitive detection of soy (Glycine max) by real-time polymerase chain reaction targeting the mitochondrial atpA gene

Detection of trace amounts of allergens is essential for correct labeling of food products by the food industry. PCR-based detection methods currently used for this purpose are targeting sequences of DNA present in the cell nucleus. In addition to nuclear DNA, a substantial amount of mitochondrial DNA (mtDNA) copies are present in the cytoplasm of eukaryotic cells. The nuclear DNA usually consists of a set of DNA molecules present in two copies per cell, whereas mitochondrial DNA is present in a few hundred copies per cell. Thus, an increase in sensitivity can be expected when mtDNA is used as the target. In this study, we present a reporter probe-based real-time PCR method amplifying the mitochondrial gene of the alpha chain of adenosine triphosphate synthetase from soy. Increase in sensitivity was examined by determining the minimal amount of soy DNA detectable by mtDNA and nuclear DNA (nDNA) amplification. Additionally, the LOD of soy in a food matrix was determined for mtDNA amplification and compared to the LOD determined by nDNA amplification. As food matrix, a model spice spiked with soy flour was used. Sensitivity of PCR-based soy detection can be increased by using mtDNA as the target.     

烟叶中β -胡萝卜素的分离纯化和测定

采用直接柱色谱的方法,用石油醚单一溶剂作为洗脱液,从霉烟或碎烟末浸膏中提取了β- 胡萝卜素纯品。通过 1H-NMR和 13 C-NMR谱图鉴定了β -胡萝卜素的结构。用高效液相色谱法(HPLC)测得β -胡萝卜素纯品的纯度为98%,测定方法准确性高、重现性好,β-胡萝卜素纯品的红外光谱图和熔点均与文献值相符。利用该方法提取β -胡萝卜素纯品,不仅收率达到80%,纯度好,而且单一溶剂作为洗脱液很容易回收再利用,因此说该方法是一种有价值的生产天然β-胡萝卜素的方法,具有良好的工业应用前景。     

Label-free characterisation of oligonucleotide hybridisation using reflectometric interference spectroscopy

The potential of a label-free detection method, reflectometric interference spectroscopy (RIfS), for temperature-dependent DNA hybridisation experiments (for example in single nucleotide polymorphism (SNP) analysis) is investigated. Hybridisations of DNA, peptide nucleic acid (PNA), and locked nucleic acid (LNA) to a single stranded DNA were measured for several temperatures, and the melting curves and temperatures were calculated from the changes in optical thickness obtained. These measurements were performed by hybridising surface-immobilised single stranded oligomers with their complementary ssDNA or with ssDNA containing SNPs at different temperatures. DNA was compared to its analogue oligomers PNA and LNA due to their stability against nuclease. A comparison of melting temperatures demonstrated the higher binding affinities of the DNA analogues. Moreover, a continuous melting curve was obtained by first hybridising the functionalised surface with its complementary DNA at room temperature and then heating up in-flow. Measurement of the continuous melting curve was only possible due to the insensitivity of the RIfS method towards temperature changes. This is an advantage over other label-free detection methods, which are based on determining the refractive index.Dedicated to the memory of Wilhelm Fresenius.     

DNA-bound lipids: computer modeling of DNA interaction with stearic acid and unsaturated fatty acids

It was shown for the first time by computer experiments that fatty acids are strongly bound to DNA. This is consistent with the presence of free fatty acids in the specimens of DNA-bound lipids isolated from various cells. Binding of all fatty acids to the DNA minor groove is stronger than to the major groove, which is correlated with the presence of two pools of free fatty acids isolated from DNA specimens by biochemical methods. Since DNA polymerase is also bound to the DNA minor groove, fatty acids can play an important role in the regulation mechanism of DNA replication and signal transmission. The energy of interaction of fatty acids with DNA depends on both the number of double bonds and the geometric configuration of the fatty acid and the nucleotide composition of DNA. Dependence on the bond energy in the DNA—fatty acid complex on the nucleotide composition attests to the possibility of site-specific binding of lipids to DNA. On passing from a saturated fatty acid to unsaturated acids containing one, two, or three double trans-bonds, the bond energy of DNA with the fatty acid gradually decreases. The presence of one or three double cis-bonds results in weakening of the strength of the DNA—fatty acid complexes compared to those with the saturated acid. The strongest binding between DNA and fatty acid was found for the unsaturated acid with two double cis-bonds (linoleic). This can be explained by the fact that the bent (boomerang) shape of the molecule of this acid follows the curve of the DNA helix. The pattern of variation of the energy of DNA complexes with stearic, linoleic, oleic, and linolenic acids correlates with experimental data on the melting points of these complexes: the more stable the DNA—fatty acid complex, the lower the melting point of DNA.     

Product differentiation during continuous-flow thermal gradient PCR

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.     

A study of DNA from chloroplasts separated by counter-current distribution.

Three types of chloroplasts (Peak I, Peak II, and Peak III chloroplasts) isolated by counter-current distribution in aqueous polymer two-phase systems have been studied with respect to DNA content. (The characterization was performed by studying the buoyant density, the melting profile and the amount of DNA of each type). The DNA was found to differ between the three types of chloroplasts.     

聚苯硫醚及其共混物的双熔融行为

聚苯硫醚及其共混物的双熔融行为麦堪成，章明秋，曾汉民，祁世成（中山大学材料科学研究所，广州，５１０２７５）关键词熔融行为，聚苯硫醚，共混物聚苯硫醚（ＰＰＳ）作为结晶型工程塑料，具有优异的力学性能，高的耐化学性以及良好的热稳定性．然而，结晶度高的ＰＰＳ...     

Isothermal crystallisation kinetics of poly(3-hydroxybutyrate) using step-scan DSC

The crystallisation kinetics, melting behaviour and morphology, of bacterial poly(3-hydoxybutyrate) (PHB) have been investigated by using differential scanning calorimetry (DSC), step-scan DSC (SDSC), wide angle X-ray diffraction (WAXRD) and hot stage polarised optical microscopy (HSPOM). DSC imparted isothermal crystallisation thermal history. The subsequent melting behaviour revealed that all PHB materials experienced secondary crystallisation during heating and the extent of secondary crystallisation varied depending on the crystallisation temperature. PHB samples were found to exhibit double melting behaviour due to melting of SDSC scan-induced secondary crystals, while considerable secondary crystallisation or annealing took place under the modulated heating conditions. The overall melting behaviour was rationalised in terms of recrystallisation and/or annealing of crystals. Interestingly, the PHB materials analysed by SDSC showed a broad exotherm before the melting peak in the non-reversing curve and a multiple melting peak reversing curve, verifying that the melting-recrystallisation and remelting process was operative. HSOM studies supported the conclusions from DSC that the radial growth rate of the PHB spherulites was significantly varied upon the crystallisation conditions. One form of crystals was shown by WAXRD from isothermally crystallised PHB.     

Bound water restrained by nanocellulose fibres

The higher-order structure of natural cellulose fibres changes in the presence of water. In order to investigate the effect of molecular level fibre structure, melting behaviour of water restrained by nano- and microcellulose fibre was measured by differential scanning calorimetry. Fibre size was measured by scanning electron microscopy and atomic force microscopy. It was found that the melting peak of water restrained by microcellulose fibre started at 250–260 K in a W _c (=mass of water/mass of dry sample) range from 0.5 to 1.2, whereas that of nanocellulose fibre was 230–237 K. Furthermore, peak temperature of melting of water restrained by nanocellulose was observed at around 270 K, in contrast, that of water restrained by microcellulose fibre was observed at ca. 275 K. Bound water content was calculated from melting enthalpy. Both non-freezing and freezing bound water of nanocellulose fibre was far larger than that of microcellulose. The above results suggest that a large amount of freezing bound water is restrained in nanocellulose fibres. It is thought that a larger number of isolated hydroxyl groups exist on the fibre surface.     

Influence of annealing and chain defects on the melting behaviour of poly(vinylidene fluoride)

The melting behaviour of poly(vinylidene fluoride) (PVDF) was investigated by differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering in order to study the influence of the chain defects content and of the temperature of annealing on the crystallization and melting behaviour.All the DSC scans show a double endotherm and the analysis of the data suggests that the low temperature endotherm is due to the melting of a population of thin lamellae, whose thickness increases during the annealing, but a high content of chain defects prevents the lamellar thickening and the main effect in this case is the crystallization of thin lamellae from a portion of polymer which did not crystallize during the quenching from the melt. Furthermore, the two melting endotherms, which are observed, can be partially ascribed to a melting-recrystallization process.Furthermore, stepwise isothermal cooling was performed in a differential scanning calorimeter followed by melting scans of fractionated PVDF samples to point out the possible presence of a series of endothermic peaks.     

DGEBA monomer as a solvent for syndiotactic polystyrene

Syndiotactic polystyrene (sPS) has to be processed at high temperatures (i.e. >290°C due to its melting point of 270°C), which approaches its degradation temperature. We aim to facilitate the processing of sPS by lowering its melt temperature and viscosity with a curable epoxy/amine model system as reactive solvent, which will result in a thermoplastic-thermoset polymer blend. As a first step we therefore investigated the melting behaviour of sPS in epoxy monomer, established its phase diagram, and investigated the crystalline form of sPS in these mixtures. DGEBA epoxy monomer is found to be a solvent for syndiotactic polystyrene at temperatures above 220°C. The DGEBA-sPS phase diagram was established by means of DSC, on the basis of crystallization and melting peaks. The form of the curve in the phase diagram indicates that DGEBA is a poor solvent for sPS. In WAXS studies of blends only the β crystalline form was detected, not the δ form, thus no sPS-DGEBA polymer-solvent compounds (clathrates) were detected. However, DGEBA can still serve as a monomer for improved processing as it depresses the crystallization temperature by 20 to 60 K upon addition of 20 to 90 wt% DGEBA respectively, while a 16 to 45 K melting peak depression can be observed by adding 20 to 90 wt% DGEBA.     

DNA melting in aggregates: impeded or facilitated?

How does DNA melt in columnar aggregate relative to its melting in diluted solution? Is the melting temperature increased or decreased with the aggregate density? Have DNA-DNA interactions, predominantly of electrostatic nature, an effect on the character of the melting transition? In attempt to answer these questions, we have incorporated the theory of electrostatic interactions between DNA duplexes into the simplest model of DNA melting. The analysis shows that the effect of aggregate density is very different for aggregates built of homologous (or identical) DNA fragments relative to the case of DNA with random base pair sequences. The putative attraction between homologous DNA helices hampers their melting and increases the melting temperature and can even dramatically change the character of the transition. In the aggregate of nonhomologous DNAs, the pattern of electrostatic interactions is more complicated, and their effect could be opposite; in some cases we may even expect electrostatically induced melting. These findings define new directions for melting experiments in dense DNA assemblies.     

Microfluidic thermodynamics of the shift in thermal stability of DNA duplex in a microchannel laminar flow

This paper reports the shift in thermal stability of DNA duplex and its thermodynamics spectroscopically, caused by stretching and orientation of DNA strands in a microchannel laminar flow. For direct spectroscopic measurement of the microchannel, we prepared an in-house temperature-controllable microchannel-type flow cell. The melting curves of DNA oligomers in a microchannel laminar flow were measured. For DNA oligomers with more than 10 base pairs, the melting curve shifted to the high-temperature side with higher flow speed. However, for 8-base-pair DNA oligomers, a change in the melting profile was not observed in batchwise and microchannel flows. We undertook microfluidic thermodynamic analysis to elucidate details of the shift in thermal stability of the DNA duplex in a microchannel laminar flow. Enthalpy-entropy compensation is applicable to the microfluidic thermal stability shift. We studied the relationships between the enthalpy-entropy compensation and DNA strand length or flow speed. Results showed that the enthalpy-entropy compensation was influenced by both DNA strand length and flow speed, and the penalties of enthalpy were 2-12% greater than the benefits of entropy.     

酸性条件下的DNA构象变化加速DNA变性解链

调控DNA的变性解链过程是DNA扩增与检测的关键步骤。对于传统的热循环DNA扩增策略,由于变温过程中热量分布不均一以及变温速度慢等不利因素,会直接影响DNA变性解链过程,从而降低DNA检测放大的效果、延长检测的时长。因此,探索快速、高效的调控DNA变性解链的方法具有重要的研究意义。本文发展了以胞嘧啶在酸性条件下的质子化反应为基础,通过改变溶液的pH值,来诱导DNA构象在Watson-Crick（WC）碱基对与Hoogsteen（HG）碱基对之间的分子构象转换,从而实现精准、快速、高效的DNA变性解链调控目标。结果表明,相较于传统的温控方法,pH调控方法能显著提高DNA变性的速率约6倍以上。本文发现pH调控方法通过降低双链DNA的反应焓约160 kJ/mol,从而提高双链DNA变性速率和效率。该方法具有用于DNA信号放大与检测等相关应用的潜力。     

Thermodynamically controlled separation of polyvalent 2-nm gold nanoparticle-oligonucleotide conjugates

We describe the synthesis of small (2-nm diameter) gold nanoparticles densely functionalized with thiolated DNA (DNA-Au NPs) and a method to separate these particles from excess free DNA after synthesis. The separation method utilizes the thermodynamically enhanced binding properties of 2-nm DNA-Au NPs, compared to free excess DNA, to selectively hybridize these small particles to larger (15-nm diameter) DNA-Au NPs and form aggregates that can be isolated by simple centrifugation. These 2-nm DNA-Au NPs are obtained in a 46% overall yield, have a high surface coverage of DNA (64.8 +/- 6.4 pmol/cm2), and as a result, exhibit increased melting temperatures and cooperative melting properties.     

Influence of melting conditions on the thermal behaviour of poly(l-lactic acid)

The influence of melting temperature and time on the thermal behaviour of poly(l-lactic acid) (PLLA) was studied with differential scanning calorimetry (DSC). Different melting conditions were investigated at temperature ranging from 200 to 210 °C, and for time from 2 to 20 min. For lower-molecular-weight PLLA, a single exothermic peak could be observed at cooling rate of 2 °C/min, after melted at different conditions. The obtained peak temperature and degrees of crystallinity dramatically increased with an increase of melting temperature or time. During subsequent heating scans, double melting peaks could be observed, which were significantly affected by prior melting conditions. The degradation of this material in the melt and the melt/re-crystallization mechanism might be responsible for the observations above. Apart from double melting, double cold crystallization peaks were observed during heating traces for this material after fast cooling (20 °C/min) from the melt. Prior melting conditions could significantly influence the cold crystallization behaviour. The competition between the crystallization from the nuclei remained after cooling, and that from spontaneous nucleation might be responsible for the appearance of double peaks. Additionally, the influence of melting conditions on the thermal behaviour of PLLA was dependent on the initial molecular weight.     

On the thermal behaviour of acetylphenyl-hydroxyaceticacid-4-nitrobenzyl esters

The 4-nitrobenzyl ester of acetylphenylhydroxyacetic acid differs in its melting behaviour from other nitrobenzyl esters of phenylhydroxyacetic or acetylphenylhydroxyacetic acids, the racemate having a higher melting point than the enantiomers. By means of thermal analysis, IR spectroscopy and X-ray diffractometry the ester can be shown to occur in two crystalline modifications. In the process of solidification of the molten mass, at first a modification of higher energy is formed, obviously being caused by an excess of one enantiomer, which is then exothermally rearranged in the lattice of the racemate.     

Investigation of the multiple melting behaviour of a PTT by DSC and polarized light optical microscopy

The multiple melting behaviour of isothermally crystallized bulk poly(trimethylene terephthalate) (PTT) observed using DSC has been correlated to the total depolarized light intensity (DLI) of thin films using hot-stage polarized light optical microscopy. The observation of partial melting, recrystallization and final melting in the DSC is correlated to the observation of the partial decrease, sudden increase and final decrease in DLI under the same heating conditions. Integration of real-time visible spectra of the transmitted light was used to separate the effects of retardation from pure birefringence of the colorful spherulitic thin-film PTT samples. The correlation of the results from these two methods has demonstrated clearly that the observed DSC multiple melting behaviour of this particular polymer is the illustrated effect of a process of continuous partial melting/recrystallization/final melting in the material during thermal analysis. The observed thermal behaviour of these metastable spherulitic materials is a complex function of their thermal history including crystallization temperature and anneal conditions, including scanning rate during thermal analysis.