Supercoiled Form Research Articles

DNA Damage Photoinduced by Titanium Dioxide in the Presence of Anionic Vesicles under UV Illumination: Influence of Sodium Chloride Concentration

The DNA damage caused by TiO2 under in vitro conditions by UV-A exposure in the presence of anionic vesicles of Aerosol OT (AOT) was investigated. The supercoiled form (S) in DNA plasmids was converted to the linear form (L) via the relaxed form (R). The DNA damage was slower in the presence of AOT vesicles prepared in aqueous NaCl solution. Moreover, the presence of AOT vesicles in solution after 6 h of UV irradiation was confirmed with an optical microscope. Probably, a fraction of the DNA was protected by random trapping during sonication. However, the addition of NaCl needed for the vesicle formation can decrease the TiO2 activity. On the other hand, in the absence of vesicles the NaCl concentration led to a profound influence on the adsorption of DNA onto the TiO2 surface. During UV irradiation, the degradation rate of DNA increased with increasing the salt concentration. Solutions containing vesicles were prepared at various NaCl concentrations between 10 mM and 75 mM. Consequently, the salt concentration had no significant effect on the DNA damage. The presence of NaCl can play a deleterious role during the photoinduced process. However, the encapsulation of a fraction of DNA is not excluded. In such conditions, the DNA could be protected against the reactive oxygen species.

Hydrolytic cleavage of DNA by quercetin zinc(II) complex

Quercetin zinc(II) complex was investigated focusing on its hydrolytic activity toward DNA. The complex successfully promotes the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The rate of conversion of SC to NC is 1.68 × 10 −4 s −1 at pH 7.2 in the presence of 100 μM of the complex. The hydrolytic cleavage of DNA by the complex is supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay, and T4 ligase ligation.

Hydrolytic cleavage of DNA by quercetin manganese(II) complexes

Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100μM of the complexes is found to be 1.32×10−4s−1. The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.

The photocleavage properties of a novel ruthenium(II) complex on liver cancer cells Bel-7402 DNA

A novel ruthenium(II) complex [Ru(bpy)2DMHPIP] (DMHPIP = 2-(3,5-dimethoxy-4-hydroxyphenyl)imidazo[4,5-f][1,10]phenanthroline) (1) has been synthesized and characterized by elemental analysis, 1H-NMR and ESI-MS. The DNA binding properties of the complex has also been investigated by electronic spectra and emission spectra, and the results show that complex (1) bind to DNA with high affinity. On the other hand, the photocleavage property of complex (1) on liver cancer cells lines Bel-7402 DNA has been investigated by electrophoresis agarose gels, and the results show that the complex can promote liver cancer cells Bel-7402 DNA from the supercoiled form to the nicked form excellently.

Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes

Plasmid DNA (pDNA) can occur in the compact supercoiled (SC) form, the relaxed open circular (OC) form and the linearized form. In this paper we investigated the transfection efficiency of SC, OC and linearized pDNA complexed to DOTAP/DOPE liposomes in Vero cells. Only DOTAP/DOPE liposomes containing SC pDNA showed protein expression while DOTAP/DOPE liposomes loaded with OC or linearized pDNA failed. First we questioned if the better transfection properties of the SC pDNA-containing lipoplexes could be due to a better transcription of SC pDNA in the nuclei of the cells, compared to OC and linearized pDNA. However, microinjecting (naked) SC, OC or linearized pDNA in the nuclei of the Vero cells revealed that the transcription efficiency was independent on the pDNA topology but did depend on the intranuclear concentration of the pDNA. As the amount of pDNA that reaches the nucleus is determined by the amount of pDNA that arrives in the cytosol it could be hypothesized that SC pDNA is more efficiently released from the DOTAP/DOPE liposomes when compared to OC and linearized pDNA. However, microinjecting comparable concentrations of the pDNA topologies in the cytoplasm still resulted in a significantly higher transfection in the case of SC pDNA, especially in cells that underwent cell division in the period after injection. It seems that, compared to OC and linearized pDNA, SC pDNA is better suited to reach the perinuclear region, a prerequisite to become entrapped in the nuclei of the cells during cell division.

Transferrin-mediated PEGylated nanoparticles for delivery of DNA/PLL

The purpose of this work was to determine the stability of pDNA/poly(L-lysine) complex(DNA/PLL) during microencapsulation, prepare transferrin (TF) conjugated PEGylatednanoparticles (TF-PEG-NP) loading DNA/PLL, and assess its physicochemicalcharacteristics and in vitro transfection efficiency. The DNA/PLL was prepared by mixingplasmid DNA (pDNA) in deionized water with various amounts of PLL. PEGylatednanoparticles (PEG-NP) loading DNA/PLL were prepared by a water–oil–water doubleemulsion solvent evaporation technique. TF-PEG-NP was prepared by coupling TFwith PEG-NP. The physicochemical characteristics of TF-PEG-NP and in vitrotransfection efficiency on K562 cells were measured. The results showed that free pDNAreserved its double supercoiled form (dsDNA) for only on average 25.7% aftersonification, but over 70% of dsDNA was reserved after pDNA was contracted withPLL. The particle size range of TF-PEG-NP loading DNA/PLL was 150–450 nmwith entrapment efficiency over 70%. TF-PEG-NP exhibited the low burst effect(<10%) within 1 day. After the first phase, DNA/PLL displayed a sustained release. The amountof cumulated DNA/PLL release from TF-PEG-NP with 2% polymer over 7 days was 85.4%for DNA/PLL (1:0.3 mass ratio), 59.8% and 43.1% for DNA/PLL (1:0.6) and DNA/PLL(1:1.0), respectively. To TF-PEG-NP loading DNA/PLL without chloroquine,the percentage of EGFP expressing cells was 28.9% for complexes consisting ofDNA/PLL (1:0.3), 38.5% and 39.7% for DNA/PLL (1:0.6) and DNA/PLL (1:1.0),respectively. In TF-PEG-NP loading DNA/PLL with chloroquine, more cellswere transfected, the percentage of positive cells was 37.6% (DNA/PLL, 1:0.3),47.1% (DNA/PLL, 1:0.6) and 45.8% (DNA/PLL, 1:1.0), which meant that thetransfection efficiency of pDNA was increased by over 50 times when PLL andTF-PEG-NP were jointly used as a plasmid DNA carrier, in particular, the maximalpercentage of positive cells (47.1%) from TF-PEG-NP loading DNA/PLL (1:0.6) wasabout 70 times the transfection efficiency of free plasmid DNA. The average cellviability of TF-PEG-NP loading DNA/PLL was about 90%, which meant thatTF-PEG-NP appeared to be safer than PLL alone. As a result, TF-PEG-NP loadingDNA/PLL could be a more effective non-viral vector for the delivery of pDNA.

The Two Plasmodium falciparum Nucleosome Assembly Proteins Play Distinct Roles in Histone Transport and Chromatin Assembly

The malarial parasite Plasmodium falciparum has two nucleosome assembly proteins, PfNapS and PfNapL (Chandra, B. R., Olivieri, A., Silvestrini, F., Alano, P., and Sharma, A. (2005) Mol. Biochem. Parasitol. 142, 237-247). We show that both PfNapS and PfNapL interact with histone oligomers but only PfNapS is able to deposit histones onto DNA. This property of PfNapS is divalent cation-dependent and ATP-independent. Deletion of the terminal subdomains of PfNapS abolishes its nucleosome assembly capabilities, but the truncated protein retains its ability to bind histones. Both PfNapS and PfNapL show binding to the linker histone H1 suggesting their probable role in extraction of H1 from chromatin fibers. Our data suggests distinct sites of interaction for H1 versus H3/H4 on PfNapS. We show that PfNapS and PfNapL are phosphorylated both in vivo and in vitro by casein kinase-II, and this modification is specifically inhibited by heparin. Circular dichroism, fluorescence spectroscopy, and chymotrypsin fingerprinting data together suggest that PfNapL may undergo very small and subtle structural changes upon phosphorylation. Specifically, phosphorylation of PfNapL increases its affinity 3-fold for core histones H3, H4, and for the linker histone H1. Finally, we demonstrate that PfNapS is able to extract histones from both phosphorylated and unphosphorylated PfNapL, potentially for histone deposition onto DNA. Based on these results, we suggest that the P. falciparum NapL is involved in the nucleocytoplasmic relay of histones, whereas PfNapS is likely to be an integral part of the chromatin assembly motors in the parasite nucleus.

Ruthenium(II) mixed-ligand complexes containing 2-(6-methyl-3-chromonyl)imidazo[4,5-f][1,10]-phenanthroline: Synthesis, DNA-binding and photocleavage studies

Two novel Ru(II) complexes [Ru(bpy) 2(MCMIP)] 2+ ( 1) and [Ru(phen) 2(MCMIP)] 2+ ( 2) (bpy = 2,2′-bipyridine; phen = 1,10-phenanthroline; MCMIP = 2-(6-methyl-3-chromonyl)imidazo[4,5-f][1,10]-phenanthroline) have been synthesized and characterized by elemental analysis, mass spectra and 1H NMR. The DNA-binding properties of the complexes were investigated by absorption, emission, melting temperature and viscosity measurements. Experimental results indicate that the two complexes can intercalate into DNA base pairs. Upon irradiation at 365 nm, two Ru(II) complexes were found to promote the cleavage of plasmid pBR 322 DNA from supercoiled form I to nicked form II, and the mechanisms for DNA cleavage by the complexes were also investigated.

Formation of Cu(II)–brazilin complex in the presence of DNA and its activities as chemical nuclease

Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)–brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)–brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)–brazilin complex and DNA bases.

Diffusion of isolated DNA molecules: Dependence on length and topology

The conformation and dynamics of circular polymers is a subject of considerable theoretical and experimental interest. DNA is an important example because it occurs naturally in different topological states, including linear, relaxed circular, and supercoiled circular forms. A fundamental question is how the diffusion coefficients of isolated polymers scale with molecular length and how they vary for different topologies. Here, diffusion coefficients D for relaxed circular, supercoiled, and linear DNA molecules of length L ranging from approximately 6 to 290 kbp were measured by tracking the Brownian motion of single molecules. A topology-independent scaling law D approximately L(-nu) was observed with nu(L) = 0.571 +/- 0.014, nu(C) = 0.589 +/- 0.018, and nu(S) = 0.571 +/- 0.057 for linear, relaxed circular, and supercoiled DNA, respectively, in good agreement with the scaling exponent of nu congruent with 0.588 predicted by renormalization group theory for polymers with significant excluded volume interactions. Our findings thus provide evidence in support of several theories that predict an effective diameter of DNA much greater than the Debye screening length. In addition, the measured ratio D(Circular)/D(Linear) = 1.32 +/- 0.014 was closer to the value of 1.45 predicted by using renormalization group theory than the value of 1.18 predicted by classical Kirkwood hydrodynamic theory and agreed well with a value of 1.31 predicted when incorporating a recently proposed expression for the radius of gyration of circular polymers into the Zimm model.

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768–12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8 mM HEPES, N-(2-hydroxyethyl)-piperazine- N′-2-ethanesulfonic acid, 5 mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8 mM carbonate buffer, 5 mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

A new dinuclear biphenylene bridged copper(II) complex: DNA cleavage under hydrolytic conditions

The dinucleating ligand, tpbpd (tetrapyridyl biphenylenediamine) forms a dicopper complex with practically no electronic coupling between the two copper (II) centres. The EPR spectrum of the complex is consistent with coordination of each copper ion to two nitrogens of the binuclear ligand. Cyclic voltammogram of the complex also reveals that the two copper (II) centres have identical ligating environment. This dimeric copper (II) complex is found to be a very efficient catalyst for the cleavage of plasmid DNA in the absence of any added cofactor. The amount of conversion of supercoiled form (Form I) of plasmid to the open circular form (Form II) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.5 in the presence of 150 μM of the complex is found to be 1.8 × 10 −3 s −1.

DNA nano-carriers from biodegradable cationic branched polyesters are formed by a modified solvent displacement method

DNA nano-carriers were formulated relying on biodegradable polyesters consisting of amine-modified poly(vinyl alcohol) (PVAL) backbones grafted with PLGA, based on the Marangoni effect thus avoiding detrimental shear or ultrasonic forces. These amine modified high molecular weight biodegradable polyesters combine specific characteristics, such as electrostatic interactions between DNA and cationic branched polyesters facilitating loading of NP with DNA. The resulting DNA containing NP showed hydrodynamic diameters in the range of 175–285 nm and highly positive ξ-potentials, depending on the nitrogen to phosphate (N/P) ratio used for the particle formation. Atomic force microscopy (AFM) demonstrated well-defined spherical particle morphologies. DNA was released from NP upon incubation in PBS buffer in its intact supercoiled form. Agarose gel electrophoresis demonstrated that DNA within the NP was protected from enzyme degradation. The biological efficiency of the DNA delivery by this nano-carrier was demonstrated by an in vitro transfection assay using four cell lines. Reporter gene delivery of the amine-modified polymers was higher than naked DNA (Control) and raised with increasing degree of amine substitution. Also type of amine and distance of cationic charge from the backbone play an important role. Further, this feature was shown by Luciferase expression of the pCMV-Luc plasmid with PEI 25 kDa/DNA polyplexes and NP prepared with amine modified polyesters with a grafted PLGA chain length of 10 monomers compared at equal N/P ratios. DNA loaded NP from P(68)-10 showed 8× higher transfection efficiencies than the PEI 25 kDa at an N/P ratio of 9 for both preparations. These novel DNA nano-carriers merit further investigations in particular for DNA vaccination under in vivo conditions.

Extent of Supercoiling in Plasmid DNA Vaccines

Plasmid DNA is currently being considered for immunization as an alternative approach to protein-based vaccines. The prevailing opinion is that the linear and open-circular forms of plasmid DNA are less effective than the supercoiled isoform in inducing immune responses. However, it can be argued that the plasmid is likely to be nicked and relaxed during its transport into the nucleus, regardless of the level of supercoiling present. This article investigates the validity of the recommendations regarding supercoiling requirements for optimal efficiency of DNA vaccines.Some studies appear to be consistent with the notion that cell transfection with plasmids is better with the supercoiled than the relaxed open-circular isoform. Other studies have shown that there is a minimum limit of supercoiled plasmid that needs to be present for potent in vivo responses to DNA vaccines, with the limit being reported to be >70% in one study. However, other data indicate that gene transfer is not significantly affected by the level of supercoiling of the gene, and cationic lipids and microparticles can be used to deliver both isoforms without significant loss of efficiency in vivo.To further address this issue, we evaluated the in vitro and in vivo potency of DNA plasmids encoding the HIV proteins, gp140 env and p55 gag. The in vitro results showed no relationship between the degree of supercoiling and cellular transfection efficiency. Similarly, the antibody titers measured in mice immunized with fully relaxed open-circle plasmids were not significantly different from responses elicited with supercoiled plasmid. However, linearized DNA was ineffective.Based on the findings of ourselves and others that the relaxed open-circular form of a plasmid DNA is no less efficient in transfecting cells or in eliciting antibody responses than its supercoiled form, we propose that it is possible to lower the requirement for a high percentage of supercoiled plasmid in a DNA vaccine formulation.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Assembly of Plasmid DNA into Liposomes After Condensation by Cationic Lipid in Anionic Detergent Solution

A novel strategy to prepare negatively charged and small DNA-containing liposomes after condensation of plasmid DNA by a cationic lipid in deoxycholate micelle environment is described. The average diameter of resulting complexes was 62 +/- 8 nm. DNA-containing liposomes were then prepared by dialysis. The shape of the resulting liposomes was spherical. The average diameter and the surface charge of the liposomes were 86 +/- 6 nm and -24 +/- 3 mV, respectively. The plasmid DNA inside liposomes remained in a supercoiled form after incubation with DNase.

Industrial Scale cGMP Purification of Pharmaceutical Grade Plasmid‐DNA

AbstractThe advances in gene therapy and genetic vaccination based on plasmid DNA result in an increasing demand of pharmaceutical grade plasmids produced under cGMP. A generic process consisting of a cell disintegration step followed by three different chromatography steps has been developed. Cell lysis is performed by an automated continuous reactor. The clarified lysate is further purified by hydrophobic interaction, anion exchange, size exclusion chromatography and by an ultra‐filtration step. The produced plasmid DNA was up to 98 % in the supercoiled form. The final genomic DNA content was lower than 10 μg/mg plasmid DNA, RNA was not detectable by agarose gel electrophoresis, the protein content was lower than 1 μg/mg plasmid DNA and the endotoxin content lower than 0.1 EU/mg plasmid DNA. An overall yield of 50 % and higher in the desired final buffer could be obtained. This process is scalable and does not require animal derived materials, detergents or organic solvents, meeting current standards for therapeutic applications.

Protection of DNA and membrane from gamma radiation induced damage by gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a naturally occurring plant phenol. In vitro and in vivo studies have shown that this phytochemical protected DNA and membranes against ionizing radiation. Rat liver microsomes and plasmid pBR322 DNA were exposed to various doses of gamma radiation in presence and absence of GA. Exposure of the microsomes to gamma radiation resulted in the formation of peroxides of membrane lipids measured as thiobarbituric acid reactive substances and presence of GA during irradiation prevented the formation of lipid peroxidation. Gamma irradiation of plasmid DNA resulted in induction of strand breaks in DNA resulting in disappearance of the supercoiled (ccc) form. Presence of GA during irradiation protected the DNA from undergoing the strand breaks. In in vivo studies it was found that whole body exposure of mice to gamma radiation (4 Gy) increased the formation of lipid peroxides in various tissues and damage to cellular DNA (as measured by alkaline comet assay) in peripheral blood leucocytes. Administration of GA to mice prior to whole body radiation exposure reduced the peroxidation of lipids and the damage to the cellular DNA indicating in vivo radiation protection of membranes and DNA by GA.

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.

Radiation protection by Terminalia chebula: Some mechanistic aspects

Radioprotective ability of the aqueous extract of the fruit of Terminalia chebula (TCE) was evaluated for its antioxidant and radioprotective abilities. TCE (50 microg) was able to neutralise 1,1-diphenyl-2-picrylhydrazyl, a stable free radical by 92.9%. The free radical neutralizing ability of TCE was comparable to that of ascorbate (100 microM) 93.5% and gallic acid (100 microM) 91.5% and was higher than that of the diethyldithiocarbamate (200 microM) 55.4%, suggesting the free radical activity of TCE. TCE protected the plasmid DNA pBR322 from undergoing the radiation-induced strand breaks. Radiation damage converts the supercoiled form (ccc) of plasmid to open circular form (oc); the presence of TCE during radiation exposure protected the plasmid from undergoing these damages. The administration of TCE (80 mg/kg body weight, i.p.) prior to whole body irradiation of mice (4 Gy) resulted in a reduction of peroxidation of membrane lipids in the mice liver as well as a decrease in radiation-induced damage to DNA, as assayed by single-cell gel electrophoresis (comet assay). TCE also protected the human lymphocytes from undergoing the gamma radiation-induced damage to DNA exposed in vitro to 2 Gy gamma-radiation. These results suggest the radioprotective ability of TCE.