DNA sequence-specific ligands: XIII. New dimeric Hoechst 33258 molecules,inhibitors of HIV-1 integrase in vitro

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta-or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N,N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54° relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3′-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.     

Binding of Hoechst 33258 and its Derivatives to DNA

Abstract

In the present work, we employed UV-VIS spectroscopy, fluorescence methods, and circular dichroism spectroscopy (CD) to study the interaction of dye Hoechst 33258, Hoechst 33342, and their derivatives to poly[d(AT)]·poly[d(AT)], poly(dA)·poly(dT), and DNA dodecamer with the sequence 5′-CGTATATATACG-3′. We identified three types of complexes formed by Hoechst 33258, Hoechst 33342, and methylproamine with DNA, corresponding to the binding of each drug in monomer, dimer, and tetramer forms. In a dimer complex, two dye molecules are sandwiched in the same place of the minor DNA groove. Our data show that Hoechst 33258, Hoechst 33342, and methylproamine also form complexes of the third type that reflects binding of dye associates (probably tetramers) to DNA. Substitution of a hydrogen atom in the ortho position of the phenyl ring by a methyl group has a little effect on binding of monomers to DNA. However it reduces strength of binding of tetramers to DNA. In contrast, a Hoechst derivative containing the ortho-isopropyl group in the phenyl ring exhibits a low affinity to poly(dA)·poly(dT) and poly[d(AT)]·poly[d(AT)] and binds to DNA only in the monomer form. This can be attributed to a sterical hindrance caused by the ortho-isopropyl group for side-by-side accommodation of two dye molecules in the minor groove. Our experiments show that mode of binding of Hoechst 33258 derivatives and their affinity for DNA depend on substituents in the ortho position of the phenyl ring of the dye molecule. A statistical mechanical treatment of binding of Hoechst 33258 and its derivatives to a polynucleotide lattice is described and used for determination of binding parameters of Hoechst 33258 and its derivatives to poly[d(AT)]·poly[d(AT)] and poly(dA)·poly(dT).     

Synthesis and properties of a symmetric dimeric bisbenzimidazole,a DNA-specific ligand

Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (λ_em 476 nm) within the DNA complex was synthesized. DB(5) is bound by a double-strand DNA and can differentially stain human and plant (flax) chromosomes. According to preliminary data, it provides a considerably more intensive contrast of chromosome staining than DAPI and Hoechst 33258 dyes. It was also found that DB(5) is an in vitro inhibitor of HIV-1 integrase in the reaction of 3′-processing.     

Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex

The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT > TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s(-1) to 96 s(-1). Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.     

The DNA minor groove binding agents Hoechst 33258 and 33342 enhance recombinant adeno-associated virus (rAAV) transgene expression

BACKGROUND: Recombinant adeno-associated viruses (rAAV) are commonly used in pre-clinical and clinical gene transfer studies. However, the relatively slow kinetics of rAAV transgene expression complicates in vitro and in vivo experiments. METHODS: 293 and COS-1 cells were transduced with rAAV2-EGFP, rAAV1-EGFP, or rAAV5-EGFP. The rAAV-EGFP expression was analyzed in the presence of Hoechst 33 258 or 33 342 as a function of time and concentration by flow cytometry and fluorescent microscope. Effects of Hoechst on cell cycle populations were determined by flow cytometry. Enhanced green fluorescent protein (EGFP) expression plasmids with or without AAV inverted terminal repeats (ITR) were constructed and gene expression by transient transfection was compared in the presence of Hoechst. RESULTS: We found that Hoechst 33 258 and 33 342 increase both the level and the population of EGFP gene expressing cells, transduced by several different serotypes of rAAV-EGFP. The augmentation of rAAV-EGFP expression occurs in different cell types in a concentration-dependent manner. In addition, the Hoechst 33 258 or 33 342 mediated enhancement of rAAV gene expression correlated with an increase of cells in S phase and G2/M phases of the cell cycle. Finally, gene expression from transfected ITR-containing plasmid DNA was also enhanced by Hoechst dyes. CONCLUSIONS: Our results revealed that two different, although related, DNA-binding drugs, Hoechst 33 258 and 33 342, accelerate the kinetics of rAAV transgene expression. These findings may provide the basis for more sensitive assessment of rAAV biological activity and also extend the applications of rAAV for in vivo gene transfer.     

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Abstract

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, ¹H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by ¹H NMR study. The binding constants are calculated to be 5.3?×?10⁷ and 6.8?×?10⁶ M^?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC₅₀ in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.

Communicated by Ramaswamy H. Sarma     

Ligands with affinity to specific sequence of DNA base pairs. XII. The synthesis and cytological studies of dimeric Hoechst 33258 molecules

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.     

Synthesis and anticancer activity of new thiazolo[3,2-a]pyrimidines: DNA binding and molecular modeling study

A novel series of nitrogenous heterocycles starting from chalcones including thiazolo[3,2-a]pyrimidines (20–67), were synthesized. Structure elucidation of the synthesized compounds was attained by the use of ¹H NMR, ¹³CC NMR, and Mass spectrometry. The obtained compounds were evaluated for their in vitro anticancer activity at the National Cancer Institute (NCI) 60 cell lines panel assay. Three cell lines, non-small cell lung cancer Hop-92, ovarian cancer IGROV1, and melanoma SK-MEL-2, exhibited some sensitivity against most of the tested compounds. Six compounds have passed the 5-log dose level NCI assay. Compounds 34 and 24 proved to be the most active derivatives with GI₅₀, TGI, LC₅₀ of 5.89, 20.0, 66.1% and 5.0, 19.5, 52.5% respectively. Compounds 36, 39, 63 showed lesser activity with GI₅₀, TGI, LC₅₀ 3.2, 11.8, 38.9%, 3.4, 16.6, 60.3%, 3.5, 17.8, 66.1% respectively. DNA binding assay of synthesized compound were performed. Molecular docking showed that compounds 34, 42, and 60 could effectively fit into the minor groove and selectively bind with AT base pairs. The results could confer the anticancer activity of compounds 24, 34, 36, and 39 in vitro to their abilities to bind at DNA minor groove.     

Stabilization of DNA Triple Helices Using Conjugates of Oligonucleotides and Synthetic Ligands

The possibility is discussed of stabilizing a DNA triple helix by covalent conjugation to the third strand (through its terminal phosphate) of ligands that have affinity to double and triple helices. Two types of stabilizers are considered: minor groove binders based on oligopyrroles, and triplex-specific intercalators. As a target, a synthetic 29-mer duplex containing a natural polypurine sequence of the human immunodeficiency provirus was employed. The stabilization with minor groove binders requires several conditions to be respected: a sufficiently long linker capable of reaching the minor groove from the major groove, a specific double-stranded structure of the oligopyrrole fragment, and its in-phase fitness to the target sequence. The best stabilizers of a triplex were novel conjugates in which two parallel molecules containing six pyrrole units each are linked to the same 5"-phosphate of a 16-mer triplex-forming oligonucleotide. The stabilizing properties of these derivatives were comparable to those of benzoindoloquinoline (BIQ) intercalators attached to the terminal phosphate of triple-helix forming oligonucleotides.     

DNA minor groove electrostatic potential: influence of sequence-specific transitions of the torsion angle gamma and deoxyribose conformations

The structural adjustments of the sugar-phosphate DNA backbone (switching of the γ angle (O5′–C5′–C4′–C3′) from canonical to alternative conformations and/or C2′-endo → C3′-endo transition of deoxyribose) lead to the sequence-specific changes in accessible surface area of both polar and non-polar atoms of the grooves and the polar/hydrophobic profile of the latter ones. The distribution of the minor groove electrostatic potential is likely to be changing as a result of such conformational rearrangements in sugar-phosphate DNA backbone. Our analysis of the crystal structures of the short free DNA fragments and calculation of their electrostatic potentials allowed us to determine: (1) the number of classical and alternative γ angle conformations in the free B-DNA; (2) changes in the minor groove electrostatic potential, depending on the conformation of the sugar-phosphate DNA backbone; (3) the effect of the DNA sequence on the minor groove electrostatic potential. We have demonstrated that the structural adjustments of the DNA double helix (the conformations of the sugar-phosphate backbone and the minor groove dimensions) induce changes in the distribution of the minor groove electrostatic potential and are sequence-specific. Therefore, these features of the minor groove sizes and distribution of minor groove electrostatic potential can be used as a signal for recognition of the target DNA sequence by protein in the implementation of the indirect readout mechanism.     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

Synthesis,DNA Binding,and Cleavage Studies of Co(III) Complexes with Fused Aromatic NO/NN-Containing Ligands

Four new Co(III) complexes, namely [Co(cq)₃](PF₆)₃, [Co(phen)₂(cq)](PF₆)₃, [Co(bnp)₃] (PF₆)₃, and [Co(phen)₂(bnp)](PF₆)₃ (where cq = chromeno[2,3-b]quinoline, phen = 1,10-phenanthroline and bnp = dibenzo[b,g][1,8]naphthyridine), were synthesized and structurally characterized. Spectroscopic data suggested an octahedral geometry for all the complexes. Binding studies of these complexes with double-stranded (ds)DNA were analyzed by absorption spectra, viscosity, and thermal denaturation studies. The results revealed that the metal complex intercalates into the DNA base stack as intercalator. The oxidative cleavage activities of the complexes were studied with supercoiled pUC19 DNA using gel electrophoresis and the results show that the complexes have potent nuclease activity.     

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication‐Associated Proteins

It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell‐free extracts, DNA primer extension using a structured single‐stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA‐PK complex (DNA‐PKcs and the Ku heterodimer) recognizes, and is activated by, structured single‐stranded DNA is provided. Hijacking the DNA‐PK complex by double‐stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA‐PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non‐Homologous End‐Joining (NHEJ) pathway, especially the XRCC4‐DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA‐PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA‐PK complex interferes with replication fork dynamics in vivo.     

花生种子萌发早期胚轴DNA合成与种子活力的关系

随着花生种子萌发率和活力指数的下降,胚轴DNA开始合成的时间推迟,其合成水平也降低。但DNA合成都是先于吸胀的12h(高活力胚)或18h(低活力胚)出现一个峰,然后再持续上升。腐胺预处理明显地促进老化胚轴萌发早期(12～2dh)的DNA合成。钙离子预处理则有一定抑制作用,但两种预处理均能提高种子的活力指数,并能促进吸胀30h以后的DNA合成。     

Synthesis,DNA Binding,and Oxidative Cleavage Studies of Fe(II) and Co(III) Complexes Containing Bioactive Ligands

The two complexes containing bioactive ligands of the type and [Fe(L)] (PF₆)₂ (1) (where L = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino] methyl}naphthalene-2-ol]) and [Co(L₁L₂)] (PF₆)₃ (2) (where L₁L₂ = mixed ligand of 2-seleno-4-methylquinoline and 1,10-phenanthroline in the ratio 1:2, respectively) were synthesized and structurally characterized. The DNA binding property of the complexes with calf thymus DNA has been investigated using absorption spectra, viscosity measurements, and thermal denaturation experiments. Intrinsic binding constant K_b has been estimated at room temperature. The absorption spectral studies indicate that the complexes intercalate between the base pairs of the CT-DNA tightly with intrinsic DNA binding constant of 2.8 × 10⁵ M^?1 for (1) and 4.8 × 10⁵ M^?1 for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The oxidative cleavage activity of (1) and (2) were studied by using gel electrophoresis and the results show that complexes have potent nuclease activity.     

Trypanosoma cruzi: Interaction with Vertebrate Cells. DNA Synthesis and Growth of Intracellular Amastigotes and their Relationship to Host Cell DNA Synthesis and Growth

SYNOPSIS DNA synthesis of intracellular Trypanosoma cruzi amastigotes, following the infection of bovine embryo skeletal muscle (BESM) cells, was studied by autoradiography. After penetration, there was a prereplicative lag period (∼12 h) followed by a synchronous round of DNA synthesis which was found to be independent of parasite number/BESM cell and the host cell DNA synthesis cycle. Parasite reproduction occurred, for the first time, at ∼ 21 h postinfection. It was concluded that T. cruzi trypomastigotes are in the G₁/G, phase of their cell division cycle and that after penetration parasite reproduction occurs independent of events controlling host cell DNA synthesis and growth. The early synchronous growth of intracellular amastigotes should facilitate further studies on the biochemical events controlling trypomastigote-to-amastigote transformation and amastigote reproduction. A further application is envisaged for studies on the mode of action of drugs with trypanocidal activity.     

Isolation and Characterization of a Satellite DNA Family in Achirus lineatus (Teleostei: Pleuronectiformes: Achiridae)

Agarose gels stained with Ethidium bromide and Southern blot experiments of HindIII-digested genomic DNA of Achirus lineatus evidenced the presence of monomers and multimers of a DNA segment of about 200 bp, named here Al-HindIII sequence. No signals were observed in Southern blot experiments with genomic DNA of other flatfish species. The DNA sequencing of four recombinant clones showed that Al-HindIII sequences had 204 bp and were 63.72% AT-rich. FISH experiments using a Al-HindIII sequence as probe showed bright signals in the centromeric position of all chromosomes of A. lineatus.