Improved Fluorometric DNA Determination Based on the Interaction of the DNA/Polycation Complex with Hoechst 33258

When DNA is mixed with the cationic polyelectrolyte poly(diallyldimethyl ammonium chloride) (PDDA), the DNA/PDDA complex is formed instantaneously at room temperature. This complex is much more efficient in enhancing the fluorescence of Hoechst 33258 (H 33258) than DNA alone. Based on the interaction of H 33258 with the DNA/PDDA complex, a new fluorescence assay for DNA is described. At pH 7.3 in Tris-HCl buffered solution, the DNA/PDDA complex causes a sharp enhancement in fluorescence intensity of H 33258. Simultanously, the emission maximum wavelength of H 33258 blueshifts from 490nm to 450nm, while the excitation redshifts from 345 to 350nm. The calibration graphs for calf thymus DNA (ctDNA) and herring sperm DNA (hsDNA) are both linear up to 5.0µgmL^–1 when the concentration of H 33258 and PDDA are fixed at 1.5×10^–6 and 1.6×10^–5molL^–1, respectively. The method is specific for native DNA. The 3 detection limits for ctDNA and hsDNA are 1.8 and 5.6ngmL^–1, respectively, i.e. much lower than in the presence of H 33258 alone. Four synthetic samples were determined satisfactorily. This method can also be developed to investigate the formation and the nature of the complexes between DNA and polycations, which have recently been widely applied in some fields such as genetic engineering and gene therapy.     

Interaction with plasmid DNA of Hoechst-TACN conjugates

ABSTRACT

A new family of conjugates between the Hoechst minor groove binder and the TACN metal ion ligand connected through hydrophobic alkyl or more hydrophilic oxyethyl linkers of different length has been prepared. The linkers are connected to the convex side of the Hoechst skeleton thus forcing the TACN ligand to exit the minor groove and interact with the phosphate backbone of DNA. The conjugates preserve the binding mode of Hoechst with an affinity influenced by the nature of the linker, the more hydrophobic being the more efficient. Coordination of Cu(II) or Zn(II) poorly affect these parameters. Nevertheless, the Zn(II) complex bearing a C6 linear alkyl linker induced a modest but reproducible acceleration of the hydrolytic cleavage of DNA which can be ascribed to the ability of the conjugate to deliver the hydrolytic subunit close to the DNA phosphodiester bonds.     

Synthesis of a new Pt(II) complex containing valganciclovir drug and calf-thymus DNA interaction study using multispectroscopic methods

A new complex, [Pt(valcyte)(DMSO)Cl]Cl, in which valcyte (trade name) served as valganciclovir hydrochloride drug ([2-[(2-amino-6-oxo-3H-purin-9-yl)methoxy]-3-hydroxypropyl](2S)-2-amino-3-methylbutanoate), was synthesized and characterized by different physicochemical methods. Binding interaction of this complex with calf-thymus DNA (ct-DNA) has been investigated by multispectroscopic techniques. The complex displays significant binding properties with ct-DNA. The results of fluorescence and UV–vis absorption spectroscopy indicated that this complex interacted with ct-DNA in a groove-binding mode, and the binding constant was 3.8 × 10⁴ M^?1. Furthermore, the complex induced detectable changes in the CD spectrum of ct-DNA and slightly changed its viscosity which verified the groove-binding mode. Finally, all results indicated that Pt(II) complex interact with DNA via groove-binding mode.     

大鼠脑缺血再灌注后运动皮质细胞凋亡的体视学观察

用体视学方法分析大鼠脑缺血再灌注后大脑运动皮质细胞的凋亡情况。随机将24只SD大鼠分为缺血再灌注后6、12、24 h和对照组。采用结扎大鼠左侧颈总动脉和夹闭右侧颈总动脉15m in后放开的方法制备大鼠脑缺血再灌注模型。应用Hoechst33258荧光染色及免疫组化Bax蛋白表达来检测细     

DNA电化学传感器在DNA片段识别与测定中的应用

本文用乙基－（３－二甲基丙基）碳二亚胺盐酸盐（ＥＤＣ）和羟基丁二酰亚胺（ＮＨＳ）活化已被氧化的石墨电极,然后将单链ＤＮＡ（ｓｓＤＮＡ－１）固定在石墨电极上。运用核酸杂交技术,使具有电化学活性的染料Ｈｏｅｃｈｓｔ ３３２５８ 嵌入双链ＤＮＡ 分子（ｄｓＤＮＡ）的碱基对中,在石墨电极表面形成ｄｓＤＮＡ－Ｈｏｅｃｈｓｔ ３３２５８ 层,通过伏安法测定嵌入Ｈｏｅｃｈｓｔ ３３２５８的氧化峰电流,可以识别和测定溶液中互补的ｓｓＤＮＡ－２ 片段,ｓｓＤＮＡ－２ 的浓度在４．８×１０－ ５～１．１×１０－ ７ ｍ ｇ／ｍ Ｌ范围内,有线性关系,检测限可达６．０×１０－ ８ ｍ ｇ／ｍ Ｌ。     

Fluorescence spectroscopic study of dip coated sol-gel thin film internal environment using fluorescent probes Hoechst33258 and Pyranine

The characterization of physicochemical properties of the internal environment of sol-gel thin films is required for understanding and designing applications in optical biosensors. We have investigated the dip coated tetraethyl-orthosilicate (TEOS) derived sol-gel thin films deposited on microscopic glass cover slips using molar ratio (water or ethanol / TEOS) R=32 using fluorescence spectroscopic measurements (emission, lifetime and anisotropy) on entrapped fluorescent probes. The effect of water and/or ethanol was studied as a function of storage (60 days) using fluorescent probes Hoechst 33258 (H258) and Pyranine (PY). Distribution of fluorescent probes in thin film was studied using confocal microscope. Emission maxima of H258 entrapped thin films from sol prepared using water as solvent showed emission maximum at 503 nm indicating the presence of water like environment which did not change during storage. On the contrary, PY entrapped thin films depicted emission bands at 434 nm and 513 nm, characteristics of ethanol and water respectively, up to the first few weeks and then the band at 434 nm prevailed (60 days), suggesting heterogeneous internal environment. Thin films from sol prepared using ethanol as solvent showed presence of ethanol through out storage. Fluorescence lifetime data of these probes in both sol-gel and thin films also suggested presence of heterogeneous internal environment. Thin films prepared from sol-gel using water as solvent suggested release of ethanol in the pores during hydrolysis and condensation reaction, which were clearly indicated by PY. The effect of sodium phosphate buffer was also studied in sol-gel and thin films. The results of these measurements showed that both the probes H258 and PY could be used effectively in monitoring the physicochemical properties of internal environment of thin films and sol-gel as a function of storage.     

Synthesis of three fluorescent boronic acid sensors for tumor marker Sialyl Lewis X in cancer diagnosis

Sialyl Lewis X (sLex) is a carbohydrate that is considered not only a marker for cancer, but also an antigen associated with the malignant behavior of cancerous cells. We have synthesized three fluorescent boronic acid sensors as potential sensors for sLex. Photoinduced electron transfer by a fluorescence analyzer was used to assess sensor-sLex antigen binding. The reaction was carried out in mixed aqueous solution, and Sensor 3 was identified as showing the strongest fluorescence enhancement upon binding to sLex at 10 nM. In cell-line culture experiments, Sensor 1 was shown to label sLex expression positively for HepG2, Colo 205, and COS-7 cells, and negatively for MDA-MB-231 cells; Sensor 2 did so positively for HepG2, PLC/PRF/5, and Colo 205 cells, and negatively for MDA-MB-231 and COS7 cells; and Sensor 3 did so positively for PLC/PRF/5 and HepG2 cells, and negatively for MDA-MD-231 and COS7 cells. MTT cytotoxicity experiment results showed that the three sensors are nontoxic, and Hoechst 33258 experiments showed that no apoptosis occurred.     

Dictyostelium Extracellular Vesicles Containing Hoechst 33342 Transfer the Dye into the Nuclei of Living Cells: A Fluorescence Study

Cells of the eukaryotic unicellular microorganism Dictyostelium discoideum are constitutively resistant to vital staining of their nuclei by the DNA-specific dye Hoechst 33342. By studying the mechanisms of this resistance, we evidenced that these cells expel vesicles containing the dye for detoxification (Tatischeff et al., Cell Mol Life Sci, 54: 476-87, 1998). The question to be addressed in the present work is the potential use of these extracellular vesicles as a biological drug delivery tool, using Hoechst 33342 as a model of a DNA-targeting drug. After cell growth with or without the dye, vesicles were prepared from the cell-free growth medium by differential centrifugation, giving rise to two types of vesicles. Negative staining electron microscopy showed their large heterogeneity in size. Using fluorescence techniques, data were obtained on the dye loading and its environment inside the vesicles. By UV video-microscopy, it was demonstrated that the dye-containing vesicles were able to deliver it into the nuclei of naive Dictyostelium cells, thus overcoming their constitutive resistance to the free dye. A vesicle-mediated dye-transfer into the nuclei of living human leukaemia multidrug resistant K562r cells was also observed.     

An ab initio study of the conformational preferences of Hoechst 33258 in gas-phase and aqueous solution environments

A quantum mechanical study of the conformational preferences of Hoechst 33258, a synthetic minor groove-binding drug, has been performed in both gas-phase and aqueous solution. Gas-phase calculations were performed at the HF/6-31G(d) and MP2/6-31G(d) levels of theory, whereas calculations in the aqueous solution phase were performed using the PCM model with the 6-31G(d) basis set. The molecule was divided into three fragments, which were submitted to a systematic and detailed conformational study. The results clearly indicate that Hoechst 33258 does not adopt a planar conformation in either the gas-phase or aqueous solution. Thus, a folded conformation is not induced by binding of the molecule to DNA, but is an intrinsic property of the compound. Received: 3 March 1998 / Accepted: 29 May 1998 / Published online 19 August 1998     

Assessing chromatin condensation for epigenetics with a DNA-targeting sensor by FRET and FLIM techniques

Here we propose a fluorescent sensor,Chroma-V,consisted of a Hoechst ligand(Hoe) to target chromatin DNA and a BODIPY rotor(BDP) to sense the local viscosity that reflects chromatin condensation state.Within Chroma-V,efficient FRET process from Hoe to BDP facilitated a single-excitation ratiometric imaging of nucleus DNA under fluorescence confocal microscope,which utilized the ratio of two channels to enable an intuitive visualization of chromatin condensation state.And fluorescence lifetime imaging(FLIM) based on fluorescent signal from BDP proved to be a more accurate method to quantify the changes of chromatin condensation state under different epigenetic states,including histone acetylation regulated by deacetylase inhibitors,cell apoptosis induced by DNA-bining drugs,and the epithelialmesenchymal transition of HUVEC cells induced by TGF-β.