Open Circular Form Research Articles

Infectious Plasmid Resistance and Efflux Pump Mediated Resistance

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.

Redifferentiation of human hepatoma cells induced by green tea polyphenols

A novel approach for the treatment of cancer is the differentiation therapy in which cancer cells are induced to attain a mature phenotype when exposed to differentiation inducers. To examine the effects of polyphenols extracted from green tea, i.e. ( – )-epicatechin (EC), ( – )-epigallocatechin (EGC), ( – )-epicatechin gallate (ECG) and ( – )-epigallocatechin gallate (EGCG), on the proliferation and redifferentiation of human hepatoma cell line SMMC-7721, we measured the changes of cell growth, cell surface charge and cell morphography after treament with green tea polyphenols. It was found that the growth curve of treated cells was decreased remarkably, cell surface charge of treated cells was decreased and the microvilli on the surface of treated cells were reduced obviously. It confirmed that green tea polyphenols could reverse malignant phenotypic characteristics and induced redifferentiation of SMMC-7721 cells. The ability of green tea polyphenols to inhibit reactive oxygen species (ROS)-mediated oxidative damage of DNA was also assessedin vitro by measuring the conversion of supercoiled pBR322 plasmid DNA to the open circular and linear forms. It was found that green tea polyphenols could significantly inhibit the oxidative damage of DNA induced by a water-soluble azo initiator 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH). However, they could promote the oxidative damage of DNA induced by H2O2 and Fe2+ at high concentrations. The relationship between the anti-cancer activity and antioxidation-prooxidation activity of green tea polyphenols is discussed.

Chitosan–DNA Nanoparticles: Effect on DNA Integrity, Bacterial Transformation and Transfection Efficiency

While somatic gene therapy has the potential to treat many genetic disorders, recent clinical trials suggest that an efficient and safe delivery vehicle for successful gene therapy is lacking. The current study examines the influence of two different preparation (the solvent evaporation method and the complex coacervation method) methods on the encapsulation of a model plasmid with chitosan. The ability of different molecular weights of chitosan to form nanoparticles with a plasmid, and particulated polymers to stabilize a plasmid in a supercoiled form, were examined by agarose gel electrophoresis. Protection of encapsulated pDNA offered by these nanoparticles from nuclease attack was confirmed by assessing degradation in the presence of DNase I, and the transformation of the plasmids with incubated nanoparticles were examined by β-galactosidase assay. Model pDNA existed as a mixture of both supercoiled (84.2%) and open circular (15.8%) forms. Our results demonstrated that supercoiled forms decreased while open circular forms and fragmented linear forms increased during the preparation of formulations. F1 formulation prepared by the complex coacervation method protected the supercoiled form of pDNA effectively. There weren't any significant changes in nanoparticle size and zeta potential values at pH 5.5 for a period of 3 months, but differences in particle sizes were observed after lyophilization with a cryoprotective agent. The efficiency of nanoparticles mediated transformation to Escherichia coli cells was significantly higher than naked DNA or poly-l-lysine (PLL)–DNA polycation complexes. The transfection studies were performed in COS-7 cells. A 3-fold increase in gene expression was produced by nanoparticles as compared to the same amount of naked plasmid DNA (pDNA). These observations suggest that formulations with high molecular weight (HMW) chitosan can be an effective non-viral method of gene vector in animal studies.

DNA interactions of cobalt(III) mixed-polypyridyl complexes containing asymmetric ligands

Three novel asymmetric ligands, 3-(pyridine-2-yl)-5,6-diphenyl- as -triazine (pdtb), 3-(pyridine-2-yl)- as -triazino[5,6-f]acenaphthylene (pdta) and 3-(pyridine-2-yl)- as -triazino[5,6-f]phenanthroline (pdtp) and their cobalt(III) complexes have been synthesized and characterized. Binding of the three complexes with calf thymus DNA (CT-DNA) has been investigated by spectroscopic methods, viscosity, cyclic voltammetry, and electrophoresis measurements. The experimental results indicate that the size and shape of the intercalated ligand have a marked effect on the binding affinity of complexes to CT-DNA. Complexes 2 and 3 have also been found to promote cleavage of plasmid pBR322 DNA from the supercoiled form I to the open circular form II upon irradiation.

DNA interactions of cobalt(III) mixed-polypyridyl complexes containing asymmetric ligands

Three novel asymmetric ligands, 3-(pyridine-2-yl)-5,6-diphenyl- as-triazine (pdtb), 3-(pyridine-2-yl)- as-triazino[5,6-f]acenaphthylene (pdta) and 3-(pyridine-2-yl)- as-triazino[5,6-f]phenanthroline (pdtp) and their cobalt(III) complexes have been synthesized and characterized. Binding of the three complexes with calf thymus DNA (CT-DNA) has been investigated by spectroscopic methods, viscosity, cyclic voltammetry, and electrophoresis measurements. The experimental results indicate that the size and shape of the intercalated ligand have a marked effect on the binding affinity of complexes to CT-DNA. Complexes 2 and 3 have also been found to promote cleavage of plasmid pBR322 DNA from the supercoiled form I to the open circular form II upon irradiation.

Specificity of the Endonuclease Activity of the Baculovirus Alkaline Nuclease for Single-stranded DNA

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) likely participates in the maturation of virus genomes and in DNA recombination. AcMNPV AN was expressed in a recombinant baculovirus as a His -tagged fusion and obtained in pure form (*AN) or as a (6)complex with the baculoviral single-stranded DNA-binding protein LEF-3 (*AN/L3). Both AN preparations possessed potent 5' --> 3'-exonuclease and weak endonuclease activities. Mutant *AN(S146A)/L3 with a change from serine to alanine at position 146 in a conservative motif was impaired in both activities. This proved that the endonuclease is an intrinsic activity of baculovirus AN. The AN endonuclease showed specificity for single-stranded DNA and converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) by a single strand break. Plasmid DNA relaxed with topoisomerase I was resistant to *AN/L3 indicating that the partially single-stranded regions in negatively supercoiled molecules served as targets for the endonuclease. Unwinding the supercoiled DNA with ethidium bromide also made DNA resistant to AN/L3. In reactions with nicked circular DNA (RFII), AN and AN/L3 hydrolyzed exonucleolytically the broken strand or cut endonucleolytically the intact strand at the position opposite the nick (gap). When LEF-3 was added to the assay, the balance between the exonucleolytic and endonucleolytic modes of hydrolysis shifted in favor of the exonuclease. The data suggest that the AN endonuclease may digest the intermediates in replication and recombination at positions of structural irregularities in DNA duplexes, whereas LEF-3 may further regulate processing of the intermediates by AN via the endonuclease and exonuclease pathways.

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.

Plasmid DNA purification.

The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA. However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA. Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing. The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA. The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material. The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA. Master and working cell banks need to be characterised and established. Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms. Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised. To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform. A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i). different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii). the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii). the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins). We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy.

DNA Scission Chemistry and EPR Studies of Four New Bis(2,6-Dimethoxyhydroquinone - 3-Mercaptoacetic Acid) - Peptide Conjugates

In an effort to investigate the use of short peptide chains as carriers of new antitumour agents, four tripeptide cytotoxic agent conjugates, namely DMQ–MA–Lys(DMQ–MA)–Lys(Cbz)–Arg–OMe, DMQ–MA–Lys(DMQ–MA)–Phe–Arg–OMe, DMQ–MA–Lys(DMQ–MA)–Ile–Arg–OMe, and DMQ–MA–Lys(DMQ–MA)–Val–Arg–OMe, were synthesized. The cytotoxic agent conjugated to the N-terminal, and the ξ-amino group of lysine of the tripeptides was 2,6-dimethoxyhydroquinone–3-mercaptoacetic acid (DMQ–MA). The tripeptides were synthesized by coupling protected amino acid residues in solution according to Pfp/DCC methods (where Pfp refers to pentafluorophenol and DCC refers to N,N′-dicyclohexylcarbodiimide). DNA scission chemistry showed that these compounds were able to cleave supercoiled DNA into open-circular form in drug concentrations as low as 5–20 μM without addition of H2O2 or UV irradiation. DNA cleavage was clearly inhibited when hydroxyl radical scavengers such as glycerol and sodium benzoate were added to the reaction system. When the drugs were added to rhodamine B, the UV absorbance and fluorescence intensity of rhodamine B decreased quickly due to the degradation caused by the hydroxyl radicals. Electron paramagnetic resonance (EPR) experiments employing the spin-trap agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that the signal intensity of the hydroxyl radicals generated from these newly synthesized DMQ–MA–peptide compounds was a non-linear function of ascorbic acid concentration. Deoxyribonucleic acid binding constants of these compounds were in the 104 M–1 range. It was concluded that DNA cleavage was not related to the ability of these drugs to produce hydroxyl radicals exclusively; indeed, it was related to their ability to produce various radicals and reactive species.

Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA.

This study investigates the feasibility of using the process of spray-freeze drying (SFD) to produce DNA dry powders for non-viral gene delivery. The effect of protective agents was assessed on the stability of DNA dry powders after SFD. The process of SFD had adverse effects on the tertiary structure of DNA with the protective agents of sucrose, trehalose and mannitol. With the protection of these sugars, a band corresponding to the linear form of DNA was observed during gel electrophoresis between the supercoiled form (SC) and the open circular (OC) form. On the contrary, excess cationic condensing polyethyleneimine (PEI), in conjunction with the above sugars, had the ability to provide protection for DNA from degradation after SFD. This is indicated by the reservation in SC and OC forms of DNA during agarose gel electrophoresis. The electrostatic forces between PEI polymer and DNA are critical for providing protection against various stresses generated by the process of SFD. Furthermore, on rehydration, the particle size and zeta potential of PEI/DNA complexes at weight ratios 3:1 of SFD dry powders were well maintained. Also, no transfection activity loss of PEI/DNA complexes at weight ratios 3:1 on NIH/3T3 cells was observed for reconstituted powders as compared with untreated control solutions. These results give a better understanding of preparing stable DNA dry powders by the process of SFD.

Scaleable processes for the manufacture of therapeutic quantities of plasmid DNA.

The need for scaleable processes to manufacture therapeutic plasmid DNA (pDNA) is easy to overlook when attention is focused primarily on vector design and establishment of early clinical results. pDNA is a large molecule and has properties that are similar to those of the contaminating chromosomal DNA. These, combined with the low initial concentration of plasmids in the host cell, provide unique process challenges that require significant upfront design to establish robust manufacturing processes that can also comply with current Good Manufacturing Practice ('cGMP') and produce milligram-to-kilogram quantities of pDNA product. This review describes promising scaleable processes that are currently being assessed for production of therapeutic supercoiled pDNA. Fermentation strategies for improving supercoiled plasmid yield and reducing contaminant concentrations are reviewed, and downstream processes are assessed for their ability to efficiently remove cellular contaminants, separate the supercoiled form of the pDNA from its open circular and linear forms, and prepare the purified drug substance for formulation. Current strategies are presented for developing stable delivery systems, and approaches to quality assurance and quality control are discussed.

Nanoparticles bearing polyethyleneglycol-coupled transferrin as gene carriers: preparation and in vitro evaluation

The aims of this work were to determine the stability of pDNA against various conditions during microencapsulation, prepare transferrin (TF)-conjugated PEGylated polycyanoacrylate nanoparticles (TF–PEG-nanoparticles), and assess its physicochemical characteristics and in vitro targeting cells association. The open circular forms of pDNA obviously increased when pDNA was emulsified into organic solution under sonification. When pDNA solution (pH 7.0) contained 1, 3 or 5% (w/v) PVA, after sonification, average 48.2, 59.4 and 62.1% of double-supercoiled DNA (dsDNA) were preserved, respectively. When medium of pDNA was 0.9% NaCl (pH 7.0), 0.1 M NaHCO 3 (pH 8.0) or phosphate buffer (pH 8.0), average 53.1, 69.3 and 56.9% of dsDNA remained after sonification, respectively. Poly(aminopoly(ethylene glycol)cyanoacrylate-co-hexadecyl cyanoacrylate) (poly(H 2NPEGCA-co-HDCA)) showed a slight influence on pDNA in 0.1 M NaHCO 3 (pH 8.0) when its concentration increased from 0.5 to 4% (w/v). TF–PEG-nanoparticles loading pDNA were spherical in shape with size under 200 nm and entrapment efficiency 35–50%. 0.1 M NaHCO 3 with 3% PVA (w/v) could largely reduce the damage of pDNA during microencapsulation. TF–PEG-nanoparticles bore 1–3% of the total PEG chains conjugated to TF molecules, and exhibited the burst effect with over 30% drug release within 1 day. After the first phase, pDNA release profiles displayed a sustained release. The amount of cumulated pDNA release over 7 days was: 86.3, 81.5 and 74.4% for 1, 2 and 4% polymer nanoparticles, respectively. The degree of target K562 cell binding of TF–PEG-nanoparticles was greater than that of non-targeted PEG-nanoparticles at 4 °C. The presence of free TF decreased significantly the degree of cell binding of TF–PEG-nanoparticles, which revealed that the binding of TF–PEG-nanoparticles to K562 cells was indeed receptor specific. These results suggested that TF–PEG-nanoparticles were useful for delivery of pDNA to target cells.

Fate and transport of antibiotic resistance genes in saturated soil columns

Interest in the fate of microorganisms (genetically modified or not) added to soil for a range of applications (e.g., biological fertilizer, bioremediation) has led to the examination of the transport and dispersion of bacteria in soil and ground water environments. The fate of added microorganisms can also be related to the fate of their DNA or any DNA found within soil or groundwater. However, studies have not examined the movement and fate of plasmids themselves in water-saturated soils. Continuous-flow water-saturated soil columns were used to examine the environmental fate and movement of antibiotic resistance genes. The genes were located on a plasmid pLEP01, which was either introduced into the soil columns directly as a mixture of supercoiled and open circular forms or after linearization by restriction enzymes. Both quantitative and qualitative measurements were done throughout the study. Quantitative measurements included the total DNA and conservative tracer concentration in the column effluent and the transformation frequencies for E. coli DH 10B and Acinetobacter sp. BD 413 in the presence of column effluent. Qualitative measurements included relative degradation of the introduced DNA by gel electrophoresis and the potential of effluent DNA to transform bacteria leading to the production of antibiotic resistant bacteria. The extent of DNA degradation was found to be proportional to its residence time in the soil column. In addition, tests on the biological activity (bacterial transformation) of the DNA demonstrated that this activity was roughly proportional to the remaining DNA after its degradation. Results suggest a potential for biologically active DNA to be transported over considerable distances in water-saturated soil and ground water.

Evaluation of the stability of polymer-based plasmid DNA delivery systems after ultrasound exposure

Under ultrasound exposure, the stability of plasmid DNA protected by polymer-based gene delivery system is an important factor for achieving optimal transfection into cells. We have evaluated the effectiveness of various polymer-based plasmid DNA delivery systems, which are interactive polymers and cationic polymers, to avoid shear degradation induced by ultrasound exposure. Alternatively, it is shown that sonication of plasmid DNA for exposure time as low as 10 s resulted in total DNA fragmentation and the loss of transfection potency in NIH/3T3 cells. Among these polymer-based plasmid DNA delivery systems, only cationic polymers had the ability to provide the protection of plasmid DNA from ultrasonic degradation as indicated by the reservation in supercoiled circular (SC) and open circular (OC) forms of plasmid DNA on the agarose gel electrophoresis. The DNA stability protected by cationic polymers decreased after ultrasound exposure in 1 M sodium chloride solution. Also, higher molecular weight of cationic polymers and sufficient cationic polymer/DNA weight ratios are essential to prevent DNA from degradation under ultrasound exposure in aqueous or salt solution. These results suggest that the protective mechanism by cationic polymers is due to the attractive bonding between cationic polymer and negative plasmid DNA. Whereas, DNA condensation alone provoked by the addition of polyethylene glycols was not sufficient to resist the DNA fragmentation induced by ultrasound exposure.

The effect of protective agents on the stability of plasmid DNA by the process of spray-drying.

The effect of several protective agents was assessed on the stability of spray-dried plasmid DNA. The spray-drying process had adverse effects on the tertiary structure of plasmid DNA with the protective agents of sucrose, glycine and agarose. With the protection of these noncondensing agents, a band corresponding to the linear form of plasmid DNA was observed in the gel electrophoresis between the supercoiled circular (SC) form and the open circular (OC) form. On the contrary, spray-dried plasmid DNA maintained some degree of structural integrity under the protection of condensing agents. For the protection by neutral condensing polymers, such as polyethylene glycol 1000 and 4000, no linear form between the SC form and the OC form of plasmid DNA was revealed in the gel electrophoresis. Also, excess cationic condensing polymer, polyethyleneimine, had the ability to provide the plasmid DNA with protection from degradation as indicated by the preservation in SC and OC forms of plasmid DNA on the agarose gel electrophoresis. Moreover, DNA topology was unchanged after six-month storage at 4 degrees C by the protection of these neutral and cationic condensing agents. Accordingly, DNA condensation induced by condensing agents may provide a way to minimize damage to plasmid DNA by the process of spray drying.

Ruthenium(ii) complexes containing asymmetric ligands: synthesis, characterization, crystal structure and DNA-binding

Two new asymmetric ligands, 3-(pyrazin-2-yl)-as-triazino[5,6-f]acenaphthylene (dta) and 3-(pyrazin-2-yl)-as-triazino[5,6-f]phenanthrene (dpt) and three novel ruthenium(II) complexes [Ru(bpy)2(L)][ClO4]2 (bpy = 2,2′-bipyridine, L = ddt (1), dta (2) and dpt (3), ddt = 3-(pyrazin-2-yl)-5,6-diphenyl-as-triazine) have been synthesized and characterized by mass spectroscopy, 1H NMR and cyclic voltammetry. The crystal structures of dta and [Ru(bpy)2(ddt)][ClO4]2 (1) were also determined. Interactions of the complexes with calf thymus DNA have also been investigated by spectrophotometric and viscosity measurements. The planar extension of the intercalative ligand increases the interaction of the complex with DNA, indicating that the size and shape of the intercalated ligand have a marked effect on the strength of interaction. The circular dichroism signals of the dialysates of the racemic complexes against calf thymus DNA suggest that complexes 2 and 3 interact enantioselectively with calf thymus DNA, but not complex 1. Complexes 2 and 3 have been found to promote the cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II upon irradiation.

The Influence Of Agarose Concentration In Gels On The Electrophoretic Trapping Of Circular Dna#

The density and structure of the electrophoretic traps for circular DNA in gels were strongly influenced by the concentration of agarose used to form the gels. Agarose gels were cast in a range of concentrations from 0.25% to 2.5% (1% is 1 g/100 mL). The trapping behaviors of two DNA circles with sizes of 13 kilobase pairs (kbp) and 52.5 were studied. The electric field strength (E) for the onset of trapping, the apparent trap density, and release characteristics of circles from traps were studied using both direct current and field inversion gel electrophoresis (FIGE) experiments. Direct current mobility indicated that the onset of trapping, indicated by a reduction in velocity and increase in band smearing, occurred at significantly lower value of E in 0.25% gels compared to 1% gels or 2% gels. The critical value of E for complete immobilization of the 13 open circular form was 16 V/cm in 0.25% gels, and 20 V/cm in 1% and 2% gels. FIGE mobility measurements, done by varying the reverse times, were used to determine the time required to free a circle from a trap. The time span of the reverse pulse time (from first release to complete release) was increased in the 0.25% gels compared to the time span for the 1% gels or 2.5% gels. These results indicated that the electrophoretic traps were more heterogeneous in the lower concentration gels. FIGE experiments, in which the forward pulse times were varied, were done to determine an average distance that a circle traveled before trapping occurred. The average distances before the 13 kbp open circles were trapped (E=22 V/cm) were approximately 80 μm in 0.25% gels, 180 μm in 1% gels, and greater than 500 μm in 2.5% gels. The higher trap density and longer trap length in low concentration gels explains the experimental observations that, under some conditions, DNA circles migrate faster in higher concentration gels compared to their migration in lower concentration gels. #Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedures. Such identification does not imply recommendation by NIST, nor does it imply that the materials or equipment are necessarily the best available for the purpose.

Preparation and characterization of plasmid DNA network via both triple helix formation and photo-crosslinking

New self-assembled network of plasmid DNA was prepared via both photo-crosslinking and triple helix formations, and evaluated the conformation of DNA network by electrophoresis. Two kinds of TFO sequences were designed to form the triple helix with pUC19 at different positions. In addition to the triple helix formation, a photo-crosslinking and lectin-molecular recognition were applied to form pUC19 network. Psoralen, photoreagent, and biotin were attached to 5′ and 3′-ends of the TFO, respectively. The biotin–TFO–psoralen conjugate, pUC19, and SA were mixed in a buffer and stand at 4 ˚C. The results of electrophoresis study indicated that both triple helix formation and photo-crosslinking with pUC19 were necessary for the network formation. Therefore, this method can provide stable plasmid DNA network that may be useful to manipulate nanostructure using typical plasmid DNA conformational changes: supercoiled circular and open circular forms.© 2003 Elsevier Science Ltd. All rights reserved.

DNA-binding and cleavage studies of a novel porphyrin ruthenium mixed complex [MPyTPP-Ru(pip)2Cl

A novel porphyrin ruthenium mixed complex, [MPyTPP—Ru(pip)2Cl]+, has been prepared and characterized by elemental analyses, ES-MS, u.v.–vis. and electrochemistry. The DNA binding properties of this complex have been investigated by absorption spectroscopy, fluorescence spectroscopy and by viscosity measurements. The results indicate that the complex may bind with DNA in a groove-binding mode. Upon irradiation with u.v. light, this complex has been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled form I to the open circular form II.

Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption

Separation of the different plasmid isoforms is a major challenge in purifying plasmid DNA. We describe a new type of biochemical interaction that occurs in the presence of high concentrations of lyotropic salt and results in the selective adsorption of supercoiled plasmid DNA to aromatic thioether ligands. Under well-defined conditions, these ligands are capable of separating supercoiled plasmid DNA (ccc) from its isoform, i.e. open circular (oc) form. Integrated in a process, preceded by group separation and followed by anion-exchange chromatography, this new purification method may facilitate the production of highly purified supercoiled plasmid DNA for use in gene therapy and DNA vaccine applications.