Further evidence for redox activation of the plasmid – dirhenium(III) complexes interactions

The DNA-interactions in vitro are still necessary investigations for determination of the possible anticancer properties of the compounds, candidates for application in cancer therapy. The aim of the present work was to realize if the interaction of cis-dicarboxylates of dirhenium(III), with pivalato- (I), isobutirato- (II) and adamanthyl- (III) ligands cleaves the plasmid in the same manner and what is the influence of the ligands on this process. For experiments we used the prokaryotic plasmid which is good model to analyze DNA-cleaving ability of different substances that exists in supercoiled conformation and turns to nicked and linear forms. It was shown that gradual conversion of the supercoiled Form I to a mixture of supercoiled (Form I) and nicked (Form II) DNA takes place and increasing amounts of Form II are produced with higher concentrations of I–III under increasing of concentration that showed the DNA-cleaving abilities of all investigated dirhenium complexes. This process was taking place with different intensity in the range I ˃ II ˃ III, that demonstrates the influence of the organic radical on the cleaving activity of the dirhenium(III) complexes. Under hydrogen peroxide conditions, I and II showed close results, demonstrating more intensive process of cleaving, including formation of the linear plasmid (Form III) under higher concentration, witnessing about redox-activation of the DNA-cleaving reaction. Cleaving activity of III was approximately the same in all experiments, that was demonstrated only by decreasing of the supercoiled form I and increasing of the nicked form II of the plasmid and by absolutely absence of the linear form III of the plasmid. The electrophoresis mobility shift assays showed that rhenium cluster compounds have nuclease activity and confirmed that natural DNA may be their target in the living cells. The conclusion was made that the mechanism of DNA-cleavage reaction of the dirhenium(III) complexes is multiple in which the electron donating (withdrawing) effects of the ligands and catalytic activity of the metal core should be taken in consideration.

Structure of the native supercoiled flagellar hook as a universal joint

Bacteria swim in viscous liquid environments by using the flagellum1–3. The flagellum is composed of about 30 different proteins and can be roughly divided into three parts: the basal body, the hook and the filament. The basal body acts as a rotary motor powered by ion motive force across the cytoplasmic membrane as well as a protein export apparatus to construct the axial structure of the flagellum. The filament is as a helical propeller, and it is a supercoiled form of a helical tubular assembly consisting of a few tens of thousands of flagellin molecules4. The hook is a relatively short axial segment working as a universal joint connecting the basal body and the filament for smooth transmission of motor torque to the filament5,6. The structure of hook has been studied by combining X-ray crystal structure of a core fragment of hook protein FlgE and electron cryomicroscopy (cryoEM) helical image analysis of the polyhook in the straight form and has given a deep insight into the universal joint mechanism7. However, the supercoiled structure of the hook was an approximate model based on the atomic model of the straight hook without its inner core domain7 and EM observations of supercoiled polyhook by freeze-dry and Pt/Pd shadow cast8. Here we report the native supercoiled hook structure at 3.1 Å resolution by cryoEM single particle image analysis of the polyhook. The atomic model built on the three-dimensional (3D) density map show the actual changes in subunit conformation and intersubunit interactions upon compression and extension of the 11 protofilaments that occur during their smoke ring-like rotation and allow the hook to function as a dynamic molecular universal joint with high bending flexibility and twisting rigidity.

Hydrolytic Activity of a Neodymium(III) Complex in DNA Cleavage

A azamacrocyclic compound with carboxyl branch, 5,5,7,12,12,1-hexamethy-1,4 ,8,11-tetraazacyclo- tetradecane-N/-acetic acid(L), and its neodymium complex ware synthesized and characterized. The mode of combination of the neodymium complex with DNA was investigated by UV-vis absorption spectroscopy methods. The cutting function of the neodymium complex to supercoiled DNA was studied by gel electrophoresis method. The results show that metal complex can bind to the phosphate of DNA double helix and promote the hydrolysis of phosphodiester bond of supercoiled DNA(Form I); Supercoiled form DNA was transformed into nicked form DNA(Form II) with strong cutting effect of the macrocyclic neodymium complex; the reaction of DNA cut is completed by a hydrolysis mechanism.

High efficiency method to obtain supercoiled DNA with a commercial plasmid purification kit.

Supercoiled state corresponds to the active form for plasmid applications. The relaxed circular form of plasmids is often inactive or poorly active. To obtain significant amounts of almost fully supercoiled DNA, we modified the standard protocol of a commercially available Qiagen plasmid purification kit. Our changes led to isolation of almost 100% of the plasmids in the supercoiled state. The modified protocol was used to purify different plasmids with consistent results. The purified plasmids maintain supercoiled state for about two months. The modified protocol is very advantageous because it allows easy DNA production with high degree of supercoiled form at low cost.

Synthesis, Characterization, DNA Binding, and Photocleavage Activity of Oxorhenium (V) Complexes withα-Diimine and Quinoxaline Ligands

The complex [ReOCl3pq] (1) (where pq = 2-(2′pyridyl)quinoxaline) has been synthesized and fully characterized by UV-Vis, FTIR, 1 and 2D NMR, and cyclic voltammetry (CV). The DNA-binding properties of the complex1as well as of the compounds [ReOCl3bpy] (2), [ReOCl3phen] (3), and pq (4) were investigated by UV-spectrophotometric (melting curves), CV (cyclic voltammetry), and viscosity measurements. Experimental data suggest that complex1intercalates into the DNA base pairs. Upon irradiation, complex1was found to promote the cleavage of plasmid pBR 322 DNA from supercoiled form I to nicked form II. The mechanism of the DNA cleavage by complex1was also investigated.

Improved downstream process for the production of plasmid DNA for gene therapy.

Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.

Molecular Recognition of Metal Complexes by DNA: A Comparative Study of the Interactions of the Parent Complexes [PtCl(TERPY)]Cl and [AuCl(TERPY)]Cl2with Double Stranded DNA

The interactions of the parent complexes [AuCl(Terpy)]Cl2and [PtCl(Terpy)]Cl with DNA were analysed by various physicochemical methods. Surprisingly, these metal complexes produce different interaction patterns with DNA in spite of their profound structural similarity. Indeed, important modifications are detected in the characteristic UV-Vis bands of [PtCl(Terpy)]Cl upon addition ofct-DNA, while the spectrum of [AuCl(Terpy)]Cl2is almost unaffected. Gel electrophoresis studies confirm these findings: [PtCl(Terpy)]Cl — but not [AuCl(Terpy)]Cl2— retards significantly the mobility of the supercoiled form of the pHV14 plasmid after a short incubation time. Ultrafiltration studies indicate that the affinity of [PtCl(Terpy)]Cl forct-DNA is significantly greater than that of [AuCl(Terpy)]Cl2. On the other hand, both [AuCl(Terpy)]Cl2and [PtCl(Terpy)]Cl induce important changes in the CD spectrum ofct-DNA, at high concentration, and increase its Tmvalue. Remarkably, the analysed metal-complex/DNA interaction patterns depend critically on the incubation times. We propose that [PtCl(Terpy)]Cl quickly intercalates DNA; then, formation of coordinative bonds progressively takes place with time. At variance, [AuCl(Terpy)]Cl2first interacts electrostatically with the DNA surface, with subsequent slow formation of some coordinative bonds.

Iron(II)-mimosine Catalyzed Cleavage of DNA

Supercoiled plasmid DNA was treated in vitro with H2O2, DTT and either Fe (II), Fe (II)-EDTA or Fe (II)- mimosine. The rate of DNA break formation was followed by the conversion of the supercoiled form into relaxed-circular and linear forms. In the concentration interval of 0-4 μм Fe (II), Fe (II)-EDTA slowed-down the formation of DNA breaks, while Fe (Il)-mimosine enhanced the rate of break formation up to several times. A conclusion is drawn that this enhancement is due to the increased affinity of the Fe (Il)-mimosine complex to DNA.