水稻线粒体DNA热变性的动力学研究

作为重要的细胞器之一，线粒体有其自身的遗传系统，它不仅有其自身的基因组，也有其独立的转录、翻译系统，甚至其使用的遗传密码也与核基因不同．线粒体DNA（mtDNA）对于线粒体本身的生物发生意义重大，尽管大多数线粒体蛋白是由细胞核基因组编码，但线粒体DNA仍有10％左右的     

External magnetic field effect on viscometer efflux times or calf thymus DNA in 0.0172 M KCI solution during the thermal denaturation process

Experiments show that during the thermal DNA denaturation process an applied magnetic field of 10 kG increases the shear viscosity (measured efflux time) of calf thymus DNA in 0.0172 M KCI solution over the viscosity of the same solution at the ambient earth field (or “no” field) condition. There is also observed an increase (although slight, about 0.1°C) in the thermal denaturation temperature of the process.     

Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption

The current paper describes the design, fabrication, and testing of a micromachined submicroliter-volume polymerase chain reaction (PCR) chip with a fast thermal response and very low power consumption. The chip consists of a bulk-micromachined Si component and hot-embossed poly(methyl methacrylate)(PMMA) component. The Si component contains an integral microheater and temperature sensor on a thermally well-isolated membrane, while the PMMA component contains a submicroliter-volume PCR chamber, valves, and channels. The micro hot membrane under the submicroliter-volume chamber is a silicon oxide/silicon nitride/silicon oxide (O/N/O) diaphragm with a thickness of 1.9 microm, resulting in a very low thermal mass. In experiments, the proposed chip only required 45 mW to heat the reaction chamber to 92 degrees C, the denaturation temperature of DNA, plus the heating and cooling rates are about 80 degrees C s(-1) and 60 degrees C s(-1), respectively. We validated, from the fluorescence results from DNA stained with SYBR Green I, that the proposed chip amplified the DNA from vector clone, containing tumor suppressor gene BRCA 1 (127 base pairs at 11th exon), after 30 thermal cycles of 3 s, 5 s, and 5 s at 92 degrees C, 55 degrees C, and 72 degrees C, respectively, in a 200 nL-volume chamber. As for specificity of DNA products, owing to difficulty in analyzing the very small volume PCR results from the micro chip, we vicariously employed the larger volume PCR products after cycling with the same sustaining temperatures as with the micro chip but with much slower ramping rates (3.3 degrees C s(-1) when rising, 2.5 degrees C s(-1) when cooling) within circa 20 minutes on a commercial PCR machine and confirmed the specificity to BRCA 1 (127 base pairs) with agarose gel electrophoresis. Accordingly, the fabricated micro chip demonstrated a very low power consumption and rapid thermal response, both of which are crucial to the development of a fully integrated and battery-powered instrument for a lab-on-a-chip DNA analysis.     

Rapid assessment of the stability of DNA duplexes by impedimetric real-time monitoring of chemically induced denaturation

In this article, we report on the electronic monitoring of DNA denaturation by NaOH using electrochemical impedance spectroscopy in combination with fluorescence imaging as a reference technique. The probe DNA consisting of a 36-mer fragment was covalently immobilized on nanocrystalline-diamond electrodes and hybridized with different types of 29-mer target DNA (complementary, single-nucleotide defects at two different positions, and a non-complementary random sequence). The mathematical separation of the impedimetric signals into the time constant for NaOH exposure and the intrinsic denaturation-time constants gives clear evidence that the denaturation times reflect the intrinsic stability of the DNA duplexes. The intrinsic time constants correlate with calculated DNA-melting temperatures. The impedimetric method requires minimal instrumentation, is label-free and fast with a typical time scale of minutes and is highly reproducible. The sensor electrodes can be used repetitively. These elements suggest that the monitoring of chemically induced denaturation at room temperature is an interesting approach to measure DNA duplex stability as an alternative to thermal denaturation at elevated temperatures, used in DNA-melting experiments and single nucleotide polymorphism (SNP) analysis.     

Fe(III)-binding collagen mimetics

The synthesis and characterization of hydroxamic acid containing single-chain and TRIS-assembled (where TRIS is tris(carboxyethoxymethyl)aminomethane) collagen mimetics are reported. We have engineered an Fe(III)-binding domain by placing a hydroxamic acid group at the C termini of collagen mimetic chains composed of the Gly-Pro-NLeu sequence. The circular dichroism spectra and thermal denaturation studies show an enhancement in triple-helical thermal stability upon the addition of Fe(III) for the TRIS-assembled structure. No triple-helical structure was detected for the single-chain collagen mimetic. From the absorbance shown in the UV-vis spectra, we believe that the thermal stabilization of the triple helix is the direct result of a coordination complex between Fe(III) and the hydroxamate groups tethered to the C termini of the collagen mimetic peptide chains.     

Sensing of Double‐Stranded DNA/RNA Secondary Structures by Water Soluble Homochiral Perylene Bisimide Dyes

A broad series of homochiral perylene bisimide (PBI) dyes were synthesized that are appended with amino acids and cationic side chains at the imide positions. Self‐assembly behavior of these ionic PBIs has been studied in aqueous media by UV/Vis spectroscopy, revealing formation of excitonically coupled H‐type aggregates. The interactions of these ionic PBIs with different ds‐DNA and ds‐RNA have been explored by thermal denaturation, fluorimetric titration and circular dichroism (CD) experiments. These PBIs strongly stabilized ds‐DNA/RNA against thermal denaturation as revealed by high melting temperatures of the formed PBI/polynucleotide complexes. Fluorimetric titrations showed that these PBIs bind to ds‐DNA/RNA with high binding constants depending on the number of the positive charges in the side chains. Thus, spermine‐containing PBIs with six positive charges each showed higher binding constants (logK_s=9.2–9.8) than their dioxa analogues (logK_s=6.5–7.9) having two positive charges each. Induced circular dichroism (ICD) of PBI assemblies created within DNA/RNA grooves was observed. These ICD profiles are strongly dependent on the steric demand of the chiral substituents of the amino acid units and the secondary structure of the DNA or RNA. The observed ICD effects can be explained by non‐covalent binding of excitonically coupled PBI dimer aggregates into the minor groove of DNA and major groove of RNA which is further supported by molecular modeling studies.     

Thermomechanical analysis of merino wool yarns

Summary There is a difference in structure across the wool fibre which is usually referred to as bilateral. The endothermic denaturation doublet of keratins has been observed by different authors for a variety of keratins and measuring conditions and mainly interpreted by different theories. Merino wool yarns have been analyzed by the thermomechanical analysis and at low stress two thermal transitions before melting have been identified. These two thermal transitions are in accordance with the onset temperatures of the denaturation doublet shown by the DSC both at temperatures lower than the thermal degradation temperature determined by TG. The DSC of fibrillated fibres by abrasion showed not a denaturation doublet but just only a denaturation peak. The two transitions of the TMA and the modification of the DSC curve by abrasion seems to confirm that abrasion removes the component which denaturates at lower temperature.     

Use of Adsorptive Transfer Stripping Voltammetry for Analyzing Variations of Cytosine Methylation in DNA

The electrochemical behavior and thermal stability of double stranded oligonucleotides containing 5‐methyl‐cytosine and 7‐deaza‐guanosine as nucleotide analogues, as well as of Jurkat genomic DNAs methylated to different degree were studied by ACV and SWV and by thernal denaturation analysis. ACV and SWV combined with thermal denaturation analysis of the natural and modified oligonucleotides gave information regarding the presence of methylation and the concomitant conformational changes. ACV and SWV of Jurkat DNA mixtures methylated to different degrees revealed a decrease of the peak heights with increasing methylation, indicating an increase of structural rigidity, in agreement with the thermal denaturation data. These results verify the, possibly local, conformational changes introduced by DNA methylation. The results obtained in all cases were reproducible.     

Towards a real-time, label-free, diamond-based DNA sensor

Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 degrees C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 degrees C and 10x PCR buffer at 30 degrees C for hybridization and 0.1 M NaOH at temperatures above 40 degrees C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.     

Different thermal unfolding pathways of catalase in the presence of cationic surfactants

In this paper we have corroborated the usefulness of spectroscopic techniques, such as UV-visible, in the study and thermodynamic characterization of the thermal unfolding of catalase as a function of the concentration and alkyl chain length of n-alkyltrimethylammonium bromides (CnTAB, n = 8, 10, and 12). For this reason, a thermodynamic model was used which included experimental data corresponding to the pre- and posttransition into the observable transition. It has been found that n-alkyltrimethylammonium bromides play two opposite roles in the folding and stability of catalase. They act as a structure stabilizer at a low molar concentration and as a destabilizer at a higher concentration. The maximum of the unfolding temperature has been found to decrease with the alkyl chain. The reason for this difference has been suggested to be the side chains involved. In the presence of C8TAB and C10TAB, Gibbs energies of unfolding (DeltaG(T)) decrease with concentration, whereas for C12TAB an increase has been observed. These findings can be explained by the fact that when differences in the hydrophobic nature of the surfactants exist, different pathways of unfolding may occur. Also, the presence of surfactants has been observed to affect the cold denaturation of catalase. Thermodynamic results suggest that the thermal denaturation of catalase in the presence of n-alkyltrimethylammonium bromides is a perfect transition between two states.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Cytotoxic activity and DNA binding of naphthalimide derivatives with amino acid and dichloroacetamide functionalizations

A series of novel naphthalimide derivatives modified by amino acids and their dichloroacetamide derivatives at the 3-position have been synthesized. Their cytotoxic activities were preliminarily evaluated against Hela, A549 and K562 cells, which showed that the length of the side chains of the amino acids influenced the cytotoxic activities. Moreover, compound 7d showed a very good cytotoxic activity against A549 cells with an IC50 value of 4.78 mmol Là1. Furthermore, the UV–vis, fluorescence,and circular dichroism(CD) spectroscopies and thermal denaturation experiment indicated that compounds 6a, 6d and 7a, 7d, as DNA intercalators, exhibited binding affinities with calf-thymus DNA(Ct-DNA).     

Kinetic nature of the thermal destabilization of LHCII macroaggregates

The main light-harvesting chl a/b pigment-protein complex of photosystem II (LHCII) in isolated state forms macroaggregates with different ultrastructure and lipid content [I. Simidjiev, V. Barzda, L. Mustardy, G. Garab, Anal. Biochem. 250 (1997) 169-175]. The thermodynamic stability of highly delipidated tightly bound LHCII macroaggregates is studied by differential scanning calorimetry and fluorescence spectroscopy. The calorimetric profile of LHCII is asymmetric, the denaturation transition is taking place at around 72 degrees C. A shoulder, which overlaps with the main denaturation transition, appears around 58 degrees C. The denaturation temperature strongly depends on the scanning rate indicating the kinetic nature of the thermal destabilization of LHCII macroaggregates. The fluorescence data prove that the thermal denaturation of LHCII is an irreversible and kinetically controlled process.     

Temperature induced hyperchromism exhibited by Hoechst 33258: evidence of drug aggregation from UV-melting method

UV-thermal denaturation is a simple optical method widely employed for determination of DNA stability and interaction with ligands. Thermal denaturation of DNA and DNA-ligand complex is usually monitored at 260 nm. These data are generally presented as a function of the absorption increase of DNA alone with no consideration of the temperature dependent hyperchromism of the free ligand. Since not every ligand has absorption at 260 nm, usually this property of the ligand is ignored. Here, we report the temperature dependent hyperchromicity exhibited by Hoechst 33258 in the presence and absence of DNA. The presence of Hoechst, added to the duplex (monophasic profile, T(m)=75 degrees C) in various ratios generates a new transition at lower temperature displaying biphasic thermal transition profiles. We attributed this new transition (hyperchromic), a mere contribution from Hoechst, which might exist in aggregated forms. The extent of drug aggregation/self-association is concentration dependent. We suggest that prior to UV-melting studies the thermal dependence of the free ligand should be investigated.     

Temperature-dependent Raman spectra of collagen and DNA

Raman spectra of collagen and DNA were discussed at different temperatures. The temperature-dependence of Raman intensity was obtained in the region from -150 to 200 degrees C. Four denaturation points at 0, 40, 68 and 90 degrees C of collagen and two peaks at 38 and 82 degrees C for DNA were obtained. The wavenumbers of many vibrational modes were found to increase for lower temperature, but the peak at 1302 cm(-1) of collagen and the peak at 1101 cm(-1) of DNA showed the opposite trend. In all of the vibrational modes of DNA, the bases showed the most sensitive to different temperatures and there is a pronounced shift of bands at 70 degrees C, the starting point of denaturation.     

Anticancer activity and DNA binding property of the trimers of triphenylethylene-coumarin hybrids

Novel trimers of triphenylethylene-coumarin hybrid containing two amino side chains(5a-d and 6a-d)were designed and synthesized by the condensation of 1,3,5-benzenetricarboxylic acid with the varied amino monomeric hybrids catalyzed by HATU and DIPEA at room temperature.The extended trimeric compound 6a(R = piperidinyl) exhibited significant anti-proliferative activity against three cancer cells at IC_(50) of near 10μmol/L UV-vis,fluorescence(lifetime) and thermal denaturation exhibited that 6a had significant interaction with Ct-DNA by the intercalative mode of binding.The order of their antiproliferative activities was 6(a,d) 5(a,d) and(5-6)a (5-6)d,respectively,in accordance with that of their DNA binding properties,which suggested that the prolonged linker(six carbons) and piperidinyl group on the side chains are beneficial to DNA binding and the anti-tumor activity.     

Effect of venous wall immobilization on the thermal degradation of collagen

The results from a comparative study of the thermal denaturation of collagen in the venous walls of reference samples and samples with varicose disease are presented. Changes in the organization of collagen network of the tissue matrix are detected via thermal analysis and multiphoton microscopy with recording of the second harmonic generation (SHG). It is established that the collagen network of venous walls degrades in varicose disease. It is shown that the disordering of the tertiary structure of collagen molecules is reflected in a 40% drop in the enthalpy of protein denaturation compared to reference (ΔH _D = 12.4 ± 4.9 J/g dry residue). The disorganization of fiber structures is recorded on SHG images. It is shown that upon the hydrothermal heating of sequestered samples of venous walls, the complete degradation of the tissue network occurs at 75°C. However, it is noted that upon the mechanical immobilization of samples of both types, the stability of collagen increases and complete denaturation is observed at temperatures above 84°C. It is suggested that the number of available conformations of polypeptide chains in the random coil state falls under tension, lowering ΔS _D and raising the temperature of the denaturation of protein.     

Circular dichroism and fluorescence spectroscopy studies of the effect of cyclodextrins on the thermal stability of chicken egg white lysozyme in aqueous solution

Circular dichroism spectroscopy revealed that the thermal stability of chicken egg white lysozyme in an aqueous buffer solution is significantly lowered by the addition of 6-O-α-d-glucosyl-β-cyclodextrin (G1-β-CD), whereas it is raised by the addition of methyl α-d-glucopyranoside. The α- and γ-cyclodextrin also lowered the thermal stability, although the effects were less prominent than that of G1-β-CD. Fluorescence spectroscopy suggested that cyclodextrins include the side chains of tryptophan residues within their internal cavities to lower the thermal stability of lysozyme. The fluorescence intensity of a sample, re-cooled to 25 °C after thermal denaturation at 75 °C in the presence of G1-β-CD, was stronger than that observed for native lysozyme. The fact that the fluorescence intensity of the re-cooling sample was stronger than that of the native one indicates that G1-β-CD persists in binding to the side chains of tryptophan residues of the re-cooled lysozyme.     

Selective Stabilization of Abasic Site‐Containing DNA by Insertion of Sterically Demanding Biaryl Ligands

Biaryl derivatives that consist of one DNA‐intercalating unit and a sterically demanding component exhibit a specific behavior towards abasic site‐containing DNA (AP‐DNA) as determined by thermal DNA denaturation experiments, spectrometric titrations and CD spectroscopic analysis. Specifically, these ligands strongly stabilize AP‐DNA towards dissociation, whereas they do not or only marginally affect the melting temperature of regular duplex DNA.     

Hybridization of PNA to structured DNA targets: quadruplex invasion and the overhang effect

Peptide nucleic acid (PNA) probes have been synthesized and targeted to quadruplex DNA. UV-vis and CD spectroscopy reveal that the quadruplex structure of the thrombin binding aptamer (TBA) is disrupted at 37 degrees C by a short PNA probe. The corresponding DNA probe fails to bind to the stable secondary structure at this temperature. Thermal denaturation experiments indicate surprisingly high thermal and thermodynamic stabilities for the PNA-TBA hybrid. Our results point to the nonbonded nucleobase overhangs on the DNA as being responsible for this stability. This "overhang effect" is found for two different PNA-DNA sequences and a variety of different overhang lengths and sequences. The stabilization offered by the overhangs assists the PNA in overcoming the stable secondary structure of the DNA target, an effect which may be significant in the targeting of biological nucleic acids, which will always be much longer than the PNA probe. The ability of PNA to invade a structured DNA target expands its potential utility as an antigene agent or hybridization probe.