New asymmetric monomethine cyanine dyes for nucleic-acid labelling: absorption and fluorescence spectral characteristics

Absorption spectra and fluorescence properties of a series of newly synthesized asymmetric monomethine cyanine dyes are studied. The dyes carry one or two positive charges. They are devoid of their own fluorescence in solution and become fluorescent upon binding to nucleic acids only. The fluorescence maxima of the new dyes are localized between 530 and 650 nm. The wavelength and intensity of fluorescence are dependent on molecular structure of the dye, type of nucleic acid and the concentration of both nucleic acid and salts. Some of the dyes are capable of distinguishing between single-stranded and double-stranded (ds) polynucleotides giving fluorescence maxima localized at different wavelengths. Detection threshold for dsDNA for most of the dyes is comparable to that of ethidium bromide. The sensitivity of the dye-dsDNA complexes to NaCl concentrations show that the new dyes interact with dsDNA by both intercalation and electrostatically.     

Comparative Study of the Radiolysis of Poly(uridylic acid) and Poly(methyluridylic acid) in Nitrous Oxide-Saturated Aqueous Solutions

A comparative study of the radiolysis of poly(uridylic acid) and poly(methyluridylic acid) in aqueous solutions saturated with N₂O was performed. The radiation-chemical yields of formation of terminal phosphate groups, intact bases, 5-hydroxy-5,6-dihydrouracil, and 5-hydroxy-5,6-dihydro-3-methyluracil and the radiation-chemical yields of decomposition of these two polynucleotides were measured. Methylation was found to inhibit almost completely the formation of terminal phosphate groups and intact bases. It was suggested that the uracil-N(3)-yl radical can be a precursor of strand breaks in poly(uridylic acid).     

Intercalation of fac-[(4,4'-bpy)ReI(CO)3(dppz)]+, dppz = dipyridyl[3,2-a:2'3'-c]phenazine, in polynucleotides. On the UV-vis photophysics of the Re(I) intercalator and the redox reactions with pulse radiolysis-generated radicals

The intercalation of fac-[(4,4'-bpy)Re(I)(CO)3(dppz)]+ (dppz = dipyridyl[3,2-a:2'3'-c]phenazine) in polynucleotides, poly[dAdT]2 and poly[dGdC]2, where A = adenine, G = guanine, C = cytosine and T = thymine, is a major cause of changes in the absorption and emission spectra of the complex. A strong complex-poly[dAdT]2 interaction drives the intercalation process, which has a binding constant, Kb approximately 1.8 x 10(5) M(-1). Pulse radiolysis was used for a study of the redox reactions of e(-)(aq), C*H(2)OH and N3* radicals with the intercalated complex. These radicals exhibited more affinity for the intercalated complex than for the bases. Ligand-radical complexes, fac-[(4,4'-bpy*)Re(I)(CO)3(dppz)] and fac-[(4,4'-bpy)Re(I)(CO)3(dppz *)], were produced by e(-)(aq) and C*H(2)OH, respectively. A Re(II) species, fac-[(4,4'-bpy)Re(II)(CO)3(dppz)](2+), was produced by N3* radicals. The rate of annihilation of the ligand-radical species was second order on the concentration of ligand-radical while the disappearance of the Re(II) complex induced the oxidative cleavage of the polynucleotide strand.     

铜(Ⅱ)-邻菲咯啉-(邻菲咯啉-5,6-二酮)络合物的合成及其同脱氧核糖核酸的作用

以邻菲咯啉（phen)、邻菲咯啉－5，6－二酮（dione)为配体首次合成了高氯酸邻菲咯啉－邻菲咯啉－5，6－二酮（Ⅱ）。用荧光光谱，摩尔比，粘度，MLCT减色效应，平衡常数以及荧光能量转移研究了各合物与鱼精子DNA的结合情况，证实了该络合物与DNA存在插入作用。基于络合物对DNA能量转移造成荧光量子产率比值（Φλ/Φ320）的降低，解释了不同波长激发光下，荧光发射峰在加入DNA后产生猝灭和增强两种绝然不同的现象。     

Study on the interaction of morphine chloride with deoxyribonucleic acid by fluorescence method

The mode and mechanism of the interaction of morphine chloride, an important alkaloid compound to calf thymus deoxyribonucleic acid (ct DNA) was investigated from absorption and fluorescence titration techniques. Hypochromic effect was founded in the absorption spectra of morphine when concentration of DNA increased. The decreased fluorescence study revealed non-cooperative binding of the morphine to DNA with an affinity of 3.94x10(3)M(-1), and the stoichiometry of binding was characterized to be about one morphine molecule per nucleotide. Stern-Volmer plots at different temperatures proved that the quenching mechanism was static. Ferrocyanide quenching study showed that the magnitude of K(SV) of the bound morphine was lower than that of the free one. In addition, it was found that ionic strength could affect the binding of morphine and DNA. Fluorescence polarization and denatured DNA studies also applied strong evidences that morphine molecule was partially intercalated between every alternate base pairs of ct DNA. As observed from above experiments, intercalation was well supported as the binding mode of morphine and ct DNA.     

Structure Analysis of PVC Nanocomposites

In this paper poly(vinyl chloride)/clay nanocomposites were prepared by melt intercalation using a single screw extruder. Problems with thermal stability of these nanocomposites during compounding were largely eliminated by pre-treatment of the organoclay with plasticizer (dioctyl phthalate), which created a barrier between polymer and quaternary amine. These nanocomposite materials were analyzed with respect to their morphology. The intercalation, exfoliation, nano-phase dispersion and orientation were investigated using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and X-ray diffraction (XRD). Moreover, different types of sample preparation for these techniques were tested as well. It was found that partially intercalated and disordered structure arose in poly (vinyl chloride) composites containing sodium type of montmorillonite, while a fine dispersion of partial to nearly full exfoliation of individual montmorillonite layers in poly (vinyl chloride) matrix was observed when this clay was organically modified. Finally, the influence of different mixing time (in extruder) on nano-phase morphology was surveyed.     

LASER FLASH SPECTROSCOPY OF METHYLENE BLUE WITH NUCLEIC ACIDS

Absorption spectra, fluorescence spectra and quantum yields, triplet state absorption spectra, yields and rates of decay have been obtained for methylene blue (MB+), MB+/CT-DNA complexes and complexes of MB+ with the synthetic polynucleotides poly[d(G-C)] and poly[d(A-T)]. Strong fluorescence quenching is observed in the complexes of the dye with CT-DNA and poly[d(G-C)] concomitant with a decrease in the triplet yield. The fluorescence and triplet yield of MB+ with poly[d(A-T)] are similar to those of the free dye. The triplet decay times are increased in all three polymer/dye complexes and show a decreased sensitivity to oxygen quenching. These results are interpreted in terms of the binding of the dye to these polymers and the implications of polymer binding on the photosensitizing properties of the dye are discussed.     

Spectroscopic study of the interaction of pazelliptine with nucleic acids

The antitumor drug pazelliptine (PZE) binds to natural and synthetic DNA sequences at 100 mM NaCl, pH 7.0, as deduced from the absorption and fluorescence data. Scatchard plots constructed from the results obtained with poly(dG-dC)-poly(dG-dC) give binding constants of base pairs in the range (2–6) × 10⁵ M⁻¹. The modifications in the absorption and fluorescence spectra observed when PZE binds to various polynucleotides, namely poly(dA-dT)-poly(dA-dT), poly(dA)-poly(dT), poly(dG-dC)-poly(dG-dC) and calf thymus DNA. reveal a change in the protonation state of the drug upon binding, increasing the apparent pK_a of its 9-N nitrogen atom. The PZE excited state properties serve as a sensitive probe to distinguish between homo and hetero A-T sites as well as between AT and GC sites. Fluorescence studies reveal that energy transfer occurs from polynucleotide bases to the bound PZE chromophore, a result consistent with an intercalative mode of binding of the drug to DNA. The emission is enhanced when PZE is bound to A-T base pairs ( 30% increase of φ_F) whereas it is quenched in the vicinity of G-C base pairs ( 90% decrease of φ_F). Furthermore, the fluorescence spectrum obtained with calf thymus DNA is hardly distinguishable from that obtained with poly(dG-dC)-polu(dG-dC), suggesting a binding of PZE to G-C rich regions.     

多柔比星稀土金属离子配合物与DNA相互作用的研究

用紫外分光光度法和荧光光谱法研究了多柔比星( Adriamycin,ADM)稀土金属离子配合物(ADM-M)与DNA的相互作用.结果发现,在pH=7.0时,ADM与Eu3+、yb3+能形成稳定配合物,该配合物可使DNA的最大吸收产生明显的减色效应及红移,并能够竞争置换溴化乙锭(EB)与DNA的结合点.KI猝灭试验发现D...     

Entropy driven binding of the alkaloid chelerythrine to polyadenylic acid leads to spontaneous self-assembled structure formation

The binding thermodynamics and interaction of the putative anticancer alkaloid chelerythrine with polyadenylic acid were investigated by isothermal titration calorimetry, absorption and fluorescence spectroscopy, circular dichroism, differential scanning calorimetry and thermal melting experiments. The equilibrium binding constant was evaluated to be of the order of 10⁷ M⁻¹. Strong positive entropic and favorable enthalpic contributions to the binding were revealed. The binding affinity was enhanced within (10 to 100) mM Na⁺ concentration. Circular dichroism spectra confirmed that the increase in entropy change was caused by a strong conformational change in the RNA polynucleotide. Absorption and circular dichroism melting studies revealed that chelerythrine binding induced self-assembled duplex structure formation in poly(A) molecules resulting in a cooperative melting profile. This was further confirmed from differential scanning calorimetry data. The intercalation binding of the alkaloid involved strong energy transfer from the polynucleotide bases to the bound alkaloid molecules. The remarkably high entropy driven binding of the alkaloid induced spontaneous self-assembled structure formation in poly(A) and the associated binding affinity is the highest so far reported for a small molecule binding to poly(A).     

Characterization of the Binding of Methylene Blue to DNA by Spectroscopic Methods

ABSTRACT

Methylene blue (MB) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism and red shifts in the absorption spectra were observed when MB binds to calf thymus DNA(CT DNA), which suggested the intercalation mechanism of MB into DNA bases. Upon binding to DNA, the fluorescence from MB was efficiently quenched by the DNA bases, with no shifts in the emission maximum. The large increases in the polarization upon binding to CT DNA supported the intercalation of MB into the helix. Ferrocyanide quenching studies showed that the magnitude of K_sv of the bound MB was lower than that of the free MB. The results of competitive binding studies showed that ethidium bromide could be displaced by MB from ethidium-DNA complex. The results of all above further studies also proved the intercalation of MB into DNA base stack.     

羟基功能化离子液体与溶菌酶相互作用的研究

采用荧光光谱和紫外吸收光谱研究了羟基功能化离子液体1-(1,2-二羟基丙基)-3-甲基咪唑氯盐([2,3-dhpmim]Cl)、1-(1,2-二羟基丙基)-3-甲基咪唑四氟硼酸盐([2,3-dhpmim]BF4)、1-(1,2-二羟基丙基)-3-甲基咪唑六氟磷酸盐([2,3-dhpmim]PF6)与溶菌酶的相互作用。研究发现此3种离子液体对溶菌酶的荧光猝灭均为静态猝灭;同步荧光显示离子液体与溶菌酶肽链上的色氨酸残基作用,色氨酸残基微环境发生改变;结合常数和结合位点数按照[2,3-dhpmim]Cl、[2,3-dhpmim]BF4、[2,3-dhpmim]PF6顺序依次增大,并随温度的升高而增大。     

Simple and sensitive method for spectrofluorimetric determination of dodecyl benzene sulfonic acid sodium

The interaction of poly(diallyldimethyl ammonium chloride) (PDDA) with dodecyl benzene sulfonic acid sodium (DBS) has been studied by fluorescence spectra. The fluorescence of DBS can be greatly enhanced by addition of PDDA, owing to the interaction between PDDA and DBS. The enhancement intensity of fluorescence was proportional to the concentration of DBS over the range 2.5x10(-7) to 9.6x10(-5)molL(-1). Its detection limit is 3.5x10(-7)molL(-1). The method has high sensitivity and selectivity and was applied to the determination of trace amounts of DBS in water samples with satisfactory result.     

Binding of Ru(bpy)3(2+) and Ru(phen)3(2+) to polynucleotides and DNA: effect to added salts on the absorption and luminescence properties

The interactions of tris(2,2'-bipyridyl)ruthenium(II) chloride and tris-(1,10-phenanthroline)ruthenium(II) chloride, Ru(bpy)3Cl2 and Ru(phen)3Cl2 respectively, with nucleic acids were studied by means of absorption spectroscopy and time-resolved and steady state luminescence techniques in unbuffered aqueous solution at room temperature as a function of added salt, oxygen and the [nucleotide]/[sensitizer] ratio (N/S). The hypochromicity of the visible absorption band of Ru(ligand)3(2+) and the changes in the luminescence intensity and luminescence decay kinetics are considerably larger in the presence of double-stranded calf thymus DNA (dsDNA) than in the presence of single-stranded DNA and polynucleotides. This is suggested to be the result of partial intercalation of the ruthenium complex into the dsDNA rather than just its higher charge density with respect to ssDNA. Spectral changes in the presence of dsDNA increase with increasing N/S ratio (maximum changes reached at N/S = 10-12, half-value 3-4). This is postulated to be due to a transition from mainly electrostatic binding to a binding in which partial intercalation plays an increased role. Addition of alkali or alkaline earth salts at very low concentrations stabilizes partial intercalation whereas higher salt concentrations lead to a release of the ruthenium complex from the strand. This effect of the salt cation increases in the order Cs less than Rb less than K less than Na less than Li less than Ba less than Sr less than Ca less than Mg less than Be. For Ru(bpy)3(2+) the presence of 0.5 mM Mg(ClO4)2 or 6 mM NaClO4 are sufficient to release 50% of the ruthenium complexes which are bound to the dsDNA (N/S = 10); the corresponding half-concentrations for Ru(phen)3(2+) are 0.8 mM and 40 mM respectively. The half-concentrations for release increase with increasing N/S ratio and decrease with the ionic radius of the added salt.     

Fabrication of nanoaggregates of a triple hydrophilic block copolymer by cetyltrimethylammonium chloride binding

A triple hydrophilic block copolymer composed of poly(ethylene oxide), poly(sodium 2-acrylamido-2-methylpropanesulfonate), and poly(methacrylic acid) (PEO-PAMPS-PMAA) does not form a micelle by itself when it is dissolved in water. However, if the anionic PAMPS and/or PMAA blocks are electrically neutralized with a cationic surfactant, such as cetyltrimethylammonium chloride (CTAC), micelle-like nanoaggregates are obtained, where the core is formed by the insolubilized PAMPS and/or PMAA blocks. Formation of the nanoaggregates was confirmed by dynamic light scattering (DLS) measurements and scanning electron microscopy (SEM), while the binding of CTAC to PEO-PAMPS-PMAA was monitored by electrophoresis measurements. The aggregates were characterized by fluorescence spectroscopy as well as DLS and SEM. It was found that the nanoaggregates have a spherical structure, and the hydrodynamic diameter ranges from 125 to 193 nm depending on the concentrations of the PEO-PAMPS-PMAA and CTAC. The critical aggregate concentration is on the order of 10-4 g L-1 when the ionic blocks of PEO-PAMPS-PMAA are fully neutralized with CTAC.     

High-performance ion-exchange chromatography of oligoribonucleotides using linear and hyperbolic salt gradients

A method for separation and chain length determination of oligo- and polynucleotides by high-performance anion-exchange chromatography was developed, which allows resolution of individual fragments according to their chain length n, up to n approximately 10 by linear gradient of sodium chloride and up to n approximately 30 by an hyperbolic gradient of this salt. The hyperbolic relationship between n and the salt concentration at which elution of the fragment occurs allows determination of the degree of polymerization of oligo- and polynucleotides with unknown n. The method proposed can be used for estimation of the effective charge of nucleic acids with complex structure.     

S-异丙甲草胺与小牛胸腺DNA的相互作用

应用紫外光谱、荧光光谱、DNA热变性法以及黏度法研究了S-异丙甲草胺与小牛胸腺DNA(ctDNA)的相互作用. 结果表明, S-异丙甲草胺使ctDNA在200 nm处的吸收峰发生明显改变, 表现出红移和减色效应, 而对260 nm处的吸收峰产生影响较小, 排除了嵌插作用的可能; ctDNA对S-异丙甲草胺内源性荧光表现出很强的猝灭作用, 且随温度的升高, 其猝灭程度有所下降, 表明S-异丙甲草胺是以形成加合物的方式与ctDNA结合的, 并求得了它们在不同温度下的结合常数; 将不同离子强度条件下S-异丙甲草胺与ctDNA作用以及不同S-异丙甲草胺浓度下ctDNA的热变性温度和黏度变化的研究结果与紫外光谱和荧光光谱相结合, 可以判断S-异丙甲草胺是以沟槽作用的方式与ctDNA结合的.     

Synthesis and photophysical evaluation of a pyridinium 4-amino-1,8-naphthalimide derivative that upon intercalation displays preference for AT-rich double-stranded DNA

The synthesis, characterisation and solid state crystal structure of a cationic 4-amino-1,8-naphthalimide derivative (1) are described. The photophysical properties of 1 are shown to vary with the solvent polarity and H-bonding ability. The fluorescence of 1 is enhanced and blue-shifted in its 1:1 complex with 5'-adenosine-monophosphate while it is partially quenched and red-shifted in its complex with 5'-guanosine-monophosphate. Linear and circular dichroism measurements show that 1 binds to double-stranded DNA by intercalation. Comparative UV-visible and fluorescence studies with double stranded synthetic polynucleotides poly(dA-dT)(2) and poly(dG-dC)(2) show that 1 binds much more strongly to the AT polymer; 1 also has a strong preference for A-T rich sequences in natural DNA. Thermal denaturation measurements also reveal a much greater stabilisation of the double-stranded poly(dA-dT)(2) than of natural DNA.     

Thermodynamic profiles of the DNA binding of benzophenanthridines sanguinarine and ethidium: A comparative study with sequence specific polynucleotides

Energetics of the binding of two known classical DNA intercalating molecules, ethidium and sanguinarine with four sequence specific polynucleotides, poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dT).poly(dA-dT), and poly(dA).poly(dT) have been compared under identical conditions. The binding of both the molecules was characterized by strong stabilization of the polynucleotides against thermal strand separation in optical melting as well as differential scanning calorimetry studies. Isothermal titration calorimetry results revealed that the binding of both sanguinarine and ethidium to poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and poly(dG).poly(dC) was exothermic and favoured by negative enthalpy changes. On the other hand, the binding of both molecules to poly(dA).poly(dT) was endothermic and entropy driven. The binding affinity values obtained from isothermal titration calorimetry data was in close proximity to that derived from thermal melting data. The heat capacity changes obtained from temperature dependence of the enthalpy change gave negative values in the range (?0.4 to 1.25) kJ · mol^?1 · K^?1 for the binding of ethidium and sanguinarine to these polynucleotides. The variations in the values indicate important differences in the formation of the complexes. New insights into the energetics and specificity aspects of interaction of these molecules to DNA have emerged from these studies.     

Surface-enhanced Raman study of the interactions between tripodal cationic polyamines and polynucleotides

Raman and surface-enhanced Raman spectra of new DNA/RNA-binding compounds consisting of three imidazole (Im) and three pyridine (Py) rings connected by tripodal polyaminomethylene linkages were obtained by the near-infrared excitation at 1064 nm. Study of interactions of Im and Py polyamines with single-stranded RNA polynucleotides (poly?A, poly?G, poly?C, poly?U), double-stranded DNA polynucleotides (poly?dAdT-poly?dAdT, poly?dGdC-poly?dGdC) and calf thymus DNA (ct-DNA) by surface-enhanced Raman spectroscopy (SERS) reveals unambiguous enhancement of the Raman scattering from the small molecules as well as appearance of new bands in spectra associated mainly with nucleobases. The SERS experiments point toward comparable interactions of Im and Py polyamines with single-stranded purine and pyrimidine polynucleotides. Furthermore, SERS experiments with double stranded polynucleotides reveal the base-pair dependent selectivity of Im and Py, whereby interactions within both, major and minor groove are indicated for poly?dAdT-poly?dAdT, at variance to preferred binding of Im and Py to only major groove of poly?dGdC-poly?dGdC. SERS spectra of Im and Py with ct-DNA imply that protonated amino groups of these compounds preferentially interact with N7 atoms (adenine, guanine) while nitrogen in aromatic rings of polyamines might be attracted to C6-NH(2) (adenine), all sites being located at the major groove of the DNA helix. Wavenumber downshift of the imidazole (Im) and pyridine (Py) ring vibrations supports aromatic stacking interactions of imidazole and pyridine aromatic moieties with DNA base-pairs.