Multiple binding modes of the camptothecin family to DNA oligomers

The binding constants of camptothecin, topotecan and its lactone ring-opened carboxylate derivative to DNA octamers were measured by UV and NMR spectroscopy. The self-association of topotecan (TPT) was also measured. The carboxylate form of TPT binds in the same way as the lactone, but more weakly. Titration of TPT into d(GCGATCGC)2 shows a preferred location stacked onto the terminal G1 base. However, the intermolecular NOEs cannot be reconciled with a single conformation of the complex, and suggest a model of a limited number of conformations in fast exchange. MD calculations on four pairs of starting structures with TPT stacked onto the G1-C8 base pair in different orientations were therefore performed. The use of selected experimental "docking" restraints yielded ten MD trajectories covering a wide conformational space. From a combination of calculated free energies, NOEs and chemical shifts, some of the structures produced could be eliminated, and it is concluded that the data are consistent with two major families of conformations in fast exchange. One of these is the conformation found in a crystal of a TPT/DNA/topoisomerase I ternary complex [Proc. Natl. Acad. Sci. USA 2002, 99, 15 387-15 392].     

The dynamics of water at DNA interfaces: computational studies of Hoechst 33258 bound to DNA

Together, spectroscopy combined with computational studies that relate directly to the experimental measurements have the potential to provide unprecedented insight into the dynamics of important biological processes. Recent time-resolved fluorescence experiments have shown that the time scales for collective reorganization at the interface of proteins and DNA with water are more than an order of magnitude slower than in bulk aqueous solution. The molecular interpretation of this change in the collective response is somewhat controversial some attribute the slower reorganization to dramatically retarded water motion, while others describe rapid water dynamics combined with a slower biomolecular response. To connect directly to solvation dynamics experiments of the fluorescent probe Hoechst 33258 (H33258) bound to DNA, we have generated 770 ns of molecular dynamics (MD) simulations and calculated the equilibrium and nonequilibrium solvation response to excitation of the probe. The calculated time scales for the solvation response of H33258 free in solution (0.17 and 1.4 ps) and bound to DNA (1.5 and 20 ps) are highly consistent with experiment (0.2 and 1.2 ps, 1.4 and 19 ps, respectively). Decomposition of the calculated response revealed that water solvating the probe bound to DNA was still relatively mobile, only slowing by a factor of 2-3, while DNA motion was responsible for the long-time component (approximately 20 ps).     

基于Hoechst33258荧光染料检测单链DNA的方法研究

根据Hoechst与ssDNA作用时不产生荧光或荧光很弱,而与dsDNA作用时荧光增强的原理,将待测ssDNA与互补ssDNA杂交形成dsDNA,实现Hoechst染料对ssDNA的检测.文中研究了不同序列、不同长度、互补链及碱基错配链等杂交产物与Hoechst染料作用的荧光强度的变化规律.同时以H1N1禽流感病毒DN...     

Unique cooperative binding interaction observed between a minor groove binding Pt antitumor agent and Hoechst dye 33258

The trinuclear compound [{Pt(NH3)3}2mu-{trans-Pt(NH3)2(H2N(CH2)6NH2)}2(6+) (0,0,0/t,t,t) binds to DNA only through noncovalent hydrogen bonding and electrostatic interactions. The presence of this 6+ cation allows discrimination of binding modes for common DNA ligands: binding of minor-groove agents such as Hoechst 33258 is cooperative, and dye-DNA interaction is enhanced whereas intercalation as exemplified by ethidium bromide is competitively inhibited.     

Multiple DNA binding modes of a metallointercalator revealed by DNA film voltammetry

Binding and the redox reaction of the metallointercalator Ru(bpy)2(dppz)2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) with DNA was investigated by DNA film voltammetry. Calf-thymus DNA (CT-DNA) was assembled on a tin-doped indium oxide electrode by layer-by-layer electrostatic adsorption. Voltammetry of Ru(bpy)2(dppz)2+ (Ru-dppz) bound to the DNA film was measured in a redox-free electrolyte and showed strong dependence on the concentration of the metallointercalator. At low Ru-dppz concentrations, a single oxidation peak was observed, the potential of which shifted from 1.25 to 1.1 V with increasing Ru-dppz concentration (peak 1). At high metal chelate concentrations, an additional oxidation peak emerged with a potential of 1.25 V which was unaffected by the Ru-dppz concentration (peak 2). Three experiments were performed to investigate the mechanism and structural basis of the multiple peaks. First, voltammetry of Os(bpy)2(dppz)2+ bound to the CT-DNA film displayed only one peak at its oxidation potential of about 0.75 V. Second, the concentration dependence of Ru-dppz bound to a poly-(AU) film (which does not contain any guanine bases) exhibited only one oxidation peak at about 1.22 V that was independent of the Ru-dppz concentration. Third, when the guanine concentration in a mixed film of CT-DNA and poly-(AU) was changed and the bound Ru-dppz was kept constant, a pre-peak emerged and shifted to 1.1 V with increasing guanines. Based on these results, the appearance of two peaks in the voltammetric measurements of CT-DNA was rationalized by invoking two different DNA binding modes for the Ru-dppz complex: intercalation and electrostatic association. Peak 2 arises from slow oxidation of guanines catalyzed by Ru-dppz electrostatically associated with the DNA film, since the addition of Mg2+ decreases the magnitude of peak 2. Peak 1 was not affected by Mg2+ ions, leading us to conclude that it is due to intercalated Ru-dppz. The intercalation positions the metal complex in close contact with the guanines inside DNA resulting in fast electrocatalytic reaction, giving rise to a catalytic pre-peak.     

Synthesis of mono-cationic and dicationic analogs of hoechst 33258

The synthesis of fourteen mono-cationic and six dicationic analogs of Hoechst 33258 is described. The monocationic benzimiazoles 7a-7g and the dicationic benzimidazoles 9a-9c are obtained in five steps starting from 4-acetamidobenzonitrile. The monocationic bis-benzimiazoles 12a-12g are synthesized in four steps starting from 4-amino-3-nitrobenzonitrile. The dicationic bis-benzimiazoles 17a-17c are obtained in six steps starting from 4-amino-3-nitrobenzonitrile.     

Protonation Equilibria of Hoechst 33258 in Aqueous Solution

The bis‐benzimidazole derivative Hoechst 33258 (2′‐(4‐hydroxyphenyl)‐5‐(4‐methylpiperazin‐1‐yl)‐2,5′‐bi‐1H‐benzimidazole) binds to the minor groove of DNA duplexes and is widely used as fluorescent cytological stain for DNA. The neutral compound, 1 , is amphiphilic with four basic and three acidic sites. We have determined all seven acidity constants by spectrophotometric titration to define the pH‐dependent distribution of species, from the fully protonated tetracation 1 ⁴⁺ to the fully deprotonated trianion 1 ^3?, in aqueous solution. The structures of the intermediate protonation states were assigned with the aid of density‐functional calculations. Electrostatic interaction free energies were calculated to adjust the acidity constants of the molecular subunits of 1 to their environment in the species 1 ⁴⁺ to 1 ^3?. The experimental and theoretical pK_a values agree well, but they differ substantially from previous estimates given in the literature.     

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.     

Electrochemical Studies for the Interaction of DNA with an Irreversible Redox Compound – Hoechst 33258

An electrochemical equation suitable for examining the interaction of irreversible redox compounds with DNA is established. According to the equation, diffusion coefficients of both free and binding compounds (D_f , D_b), binding constant (K) and binding site size (s) of compounds with DNA could be obtained simultaneously by nonlinear fit analysis of electrochemical data. Bis‐benzimidazole derivative (Hoechst 33258), as an irreversible redox compound, was investigated for its electrochemical behavior and the interaction with natural fish sperm DNA (fsDNA) using cyclic voltammetry, chronocoulometry, bulk electrolysis and scanning electrochemical microscope technique. A nonlinear fit analysis of the experimental data yielded: D_f=8.3×10^?5 cm² s^?1, D_b=6.0×10^?6 cm² s^?1, K=2.1×10⁸ cm³ mol^?1, s=3.9. The overall results suggest that Hoechst 33258 binds tightly to the minor groove of fsDNA and covers four base pairs.     

Visualization of complexes of Hoechst 33258 and DNA duplexes in solution by atomic force microscopy

Tertiary structure changes in DNA duplexes, induced by Hoechst 33258 binding, have been examined by the use of atomic force microscopy. Besides minor groove binding, which is an established mode of binding for this drug, Hoechst 33258 has now been found to show another binding mode, which causes an unwinding of the duplex. When the drug concentration is as high as 0.5 microg/ml, the Hoechst 33258 molecule seems to function as a clamp for two DNA chains and forms a condensate. The condensate was found to have a toroidal shape. By surveying more than 100 microscopic images of such condensates formed in I microg/ml drug solution, a mechanism of toroidal condensate formation has been proposed.     

Synthesis of Analogs of Hoechst 33258 Designed for Altered Base and Sequence Recognition

The syntheses of various analogs of the minor groove binding agent Hoechst 33258 I are described to explore their potential of selective helicase blockade and anticancer activity. The target compounds II a,b,c and III a,b,c were obtained by condensation of the appropriate functionalized ortho-diamines and substituted benzimidazole carboxaldehydes in nitrobenzene.     

Synthesis of Substituted Imidazo[4,5-b]pyridines as Analogues of the DNA Binding Fluorochrome Hoechst 33258

Various substituted imidazo[4,5-b]pyridine analogues of Hoechst 33258 have been synthesized to explore the potential of selective helicase blockade and anticancer activity. The target compounds II a, b & d were obtained by condensation of the appropriate functionalized diamine and substituted benzimidazole aldehydes in nitrobenzene.     

Molecular interactions between benzimide trichloride (Hoechst 33258) and DNA in dimethyl sulfoxide aqueous solutions, according to spectroscopy data

Interaction between benzimide (Hoechst 33258, H33258) and calf thymus DNA in aqueous dimethyl sulfoxide is investigated by means of UV-Vis and fluorescence spectroscopy at a constant ratio (r) of the number of H33258 molecules and DNA base pairs. Melting curves of the DNA-H33258 complex are obtained from the temperature dependences of the normalized optical density and fluorescence intensity, and the melting temperatures of the complex are determined. It is shown that adding dimethyl sulfoxide (DMSO) lowers the complex’s melting temperature. It is concluded that a long wavelength shift of the fluorescence spectra occurs when the temperature is raised.     

脱氧核糖核酸与双苯甲亚胺相互作用的荧光特性研究

采用荧光光谱法对脱氧核糖核酸（ＤＮＡ）与双苯甲亚胺（Ｈｏｃｅｈｓｔ ３３２５８）相互作用的方式及其作用机理进行了研究,证实了Ｈｏｅｃｈｓｔ ３３２５８与ＤＮＡ的相互作用除嵌入作用时,还存在持异性的静电作用;Ｈｏｅｃｈｓｔ３３２５８与ＤＮＡ主要作用在Ａ－Ｔ匹配,同时,在磷酸根的存在下可以减弱Ｈｏｅｃｈｓｔ３３２５８与ＤＮＡ的非特异性作用。另外,根据荧光峰增强与Ｈｏｅｃｈｓｔ３３２５８浓度的线性关系,     

The action spectrum (313-435 nm) for killing Hoechst 33258 treated Chinese hamster ovary cells containing bromodeoxyuridine substituted DNA

Abstract— The action spectrum (313–435 nm) for killing Chinese hamster ovary cells containing bromo-deoxyuridine substituted DNA and treated with Hoechst 33258 was very similar to the absorption spectrum of the dye bound to chromatin, indicating that sensitization was mediated through direct absorption of radiation by the dye. The ratio of sensitization cross sections for 365 nm (plus dye) to 313 nm (no dye) was approx. 30 while this ratio for strand breakage was about one. These results are in agreement with the hypothesis that strand breaks are not the major class of lethal photoproducts induced via Hoechst 33258 sensitization.     

Design of new bidentate ligands constructed of two Hoechst 33258 units for discrimination of the length of two A3T3 binding motifs

[structure: see text] The aim of this study is to develop bidentate minor-groove binders that bind the double binding motifs cooperatively. The new bidentate ligands (1) have been designed by connecting two Hoechst 33258 units with a polyether linker for cooperative binding with two remote A3T3 sites of DNA. The linker is introduced to the benzimidazole ring so that it is located at the convex side of the Hoechst unit. DNA binding affinity of the ligands was evaluated by measuring surface plasmon resonance (SPR), circular dichroism, and fluorescence spectra. Interestingly, the bidentate ligands (1) did not show affinity to DNA1 with a single A3T3 motif but showed selective affinity to DNA2 with two A3T3 motifs. The Long Bis-H (1L) having a long polyether linker showed specific binding to DNA2(6) with two A3T3 motifs separated by six nonbinding base pairs. The Long Bis-H (1L) has also shown specific binding to the three-way junction DNA4 with two A3T3 motifs. This study has demonstrated that DNA with double binding motifs can be selectively recognized by the newly designed bidentate ligands.     

Solvation dynamics of Hoechst 33258 in water: an equilibrium and nonequilibrium molecular dynamics study

Integrated within an appropriate theoretical framework, molecular dynamics (MD) simulations are a powerful tool to complement experimental studies of solvation dynamics. Together, experiment, theory, and simulation have provided substantial insight into the dynamic behavior of polar solvents. MD investigations of solvation dynamics are especially valuable when applied to the heterogeneous environments found in biological systems, where the calculated response of the environment to the electrostatic perturbation of the probe molecule can easily be decomposed by component (e.g., aqueous solvent, biomolecule, ions), greatly aiding the molecular-level interpretation of experiments. A comprehensive equilibrium and nonequilibrium MD study of the solvation dynamics of the fluorescent dye Hoechst 33258 (H33258) in aqueous solution is presented. Many fluorescent probes employed in experimental studies of solvation dynamics in biological systems, such as the DNA minor groove binder H33258, have inherently more conformational flexibility than prototypical fused-ring chromophores. The role of solute flexibility was investigated by developing a fully flexible force-field for the H33258 molecule and by simulating its solvation response. While the timescales for the total solvation response calculated using both rigid (0.16 and 1.3 ps) and flexible (0.17 and 1.4 ps) models of the probe closely matched the experimentally measured solvation response (0.2 and 1.2 ps), there were subtle differences in the response profiles, including the presence of significant oscillations for the flexible probe. A decomposition of the total response of the flexible probe revealed that the aqueous solvent was responsible for the overall decay, while the oscillations result from fluctuations in the electrostatic terms in the solute intramolecular potential energy. A comparison of equilibrium and nonequilibrium approaches for the calculation of the solvation response confirmed that the solvation dynamics of H33258 in water is well-described by linear response theory for both rigid and flexible models of the probe.     

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.     

Determination of nucleic acids based on the fluorescence quenching of Hoechst 33258 at pH 4.5

It was found the strong fluorescence emitted by the bis-benzimidazole derivative Hoechst 33258 at 490 nm could be efficiently quenched in pH 4.5 buffer when nucleic acids were added. Analysis of fluorescence intensity showed that the procedure was a static quenching dominated one, which was also demonstrated by the electron absorption spectra and lifetime of the excited state. The binding constant and numbers of binding sites were obtained from the Scatchard plot. The decreased fluorescence intensity was in proportion to the concentration of nucleic acids in the range 40-1800 ng ml⁻¹ for dsDNA and 26-1700 ng ml⁻¹ for ssDNA. The limits of detection were 12 and 8 ng ml⁻¹, respectively. The sensitivity of the method was about 3.4 times higher for dsDNA detection and 5.4 times higher for ssDNA detection compared with the widely used fluorescence enhancement method using the same dye. Application results to synthetic samples showed simplicity, rapidity and satisfactory reproducibility of the presented method. Measurement of real samples extracted from leaves of Crassula argentea and E. coli genome also gave satisfactory results, which were in good agreement with those obtained using spectrophotometric method.     

New dicationic porphyrin ligands suited for intercalation into B-form DNA

This paper describes the synthesis and characterization of a new series of sterically nondemanding, dicationic porphyrins that exhibit novel DNA-binding interactions. Cationic porphyrins continue to be the focus of a great deal of effort because of the promise they have for use in photodynamic, antiviral, and anticancer therapies. The systems explored here include 5,15-di(N-methylpyridinium-4-yl)porphyrin (H2D4), 5,15-di(N-methylpyridinium-3-yl)porphyrin (H2D3), and 5,15-di(N-methylpyridinium-2-yl)porphyrin (H2D2), as well as Zn(D4) and Zn(D3), the zinc(II)-containing derivatives of H2D4 and H2D3, respectively. Viscometry studies, in conjunction with various spectroscopic techniques, reveal the nature of the adducts formed with DNA. Irrespective of the base composition, H2D4 and H2D3 bind to DNA by intercalation. The zinc derivatives Zn(D4) and Zn(D3) are also intercalators; however, the binding constants are smaller because uptake requires the loss of an axial ligand. The decisive roles that steric factors and structural rigidity play in shaping the adducts with DNA become clear. Sequences that contain mainly adenine-thymine base pairs easily depart from the canonical B-form DNA structure and generally accommodate bulky porphyrins in external binding sites. However, with the H2D3 and H2D4 systems, the steric requirements are so minimal that intercalation becomes the preferred mode of binding, even in [poly(dA-dT)]2. The intercalated form of the H2D2 isomer is less stable, probably because of frontal strain associated with the (N-methyl)pyridinium-2-yl groups. A qualitative energy-level diagram is useful for assessing the forces that influence binding and could guide the design of new porphyrin ligands.