Supercoiled Structure Research Articles

Synthesis and characterization of latex nanoparticles using a visible-light photoinitiating system in reverse micelles

This work shows the feasibility of using the photoinitiating system composed by the xanthene dye Eosin-Y and triethanolamine on the polymerization of acrylamide in benzyl-hexadecyl-dimethylammonium chloride (BHDC) reverse micelles. Molecular weights of polyacrylamide (MWPAA) in the order of 105 and conversions up to 90 % are obtained. The size of the latex particles is in the range of the nanometers (d < 50 nm). The effect of water content and concentrations of surfactant, amine, dye, and monomer on the particle size and MWPAA are examined. Only the amine and monomer concentrations affect the MWPAA. These results are interpreted on the basis of an exchange mechanism between micelles. Practically no effect on hydrodynamic diameters is observed when the value of MWPAA is doubled. This is ascribed to a supercoiled structure of PAA inside of micelles. Our results also suggest that the polymer properties can be modulated by appropriate combination of the dye/surfactant electrical charges.

Cobalt(II) complexes with the antimicrobial drug enrofloxacin: Structure, antimicrobial activity, DNA- and albumin-binding

The cobalt(II) complexes with the quinolone antimicrobial agent enrofloxacin (Herx) in the presence of the nitrogen-donor heterocyclic ligands pyridine (py), 2,2′-bipyridylamine (bipyam), 1,10-phenanthroline (phen), 2,2′-bipyridine (bipy) or the oxygen-donor ligand N,N-dimethylformamide (DMF) were synthesized and characterized. The crystal structures of complexes [Co(erx)2(py)2]·MeOH·6H2O and [Co(erx)2(bipyam)]·4.5MeOH·1.25H2O were determined by X-ray crystallography. The deprotonated enrofloxacinato ligands are bidentately bound to cobalt(II) ion through the pyridone oxygen and a carboxylato oxygen. The antimicrobial activity of the complexes was tested against five different microorganisms (Escherichia coli XL1, Xanthomonas campestris ATCC 33013, Staphylococcus aureus ATCC 29213, Bacillus cereus ATCC 11778 and Bacillus subtilis ATCC 6633) and the complexes were more active than free Herx. The binding of the complexes to calf-thymus DNA (CT DNA) was monitored by spectroscopic techniques, viscosity measurements, cyclic voltammetry and by mobility shift experiments in agarose gel electrophoresis with CT DNA and plasmid DNA (pDNA). All complexes exhibited a preference for binding to the supercoiled structure of the pDNA. The ability of the complexes to displace ethidium bromide (EB) from the EB–DNA complex was also investigated. The experiments indicated intercalation as the most possible mode and the DNA-binding strength of the complexes were also calculated. The complexes bind to human or bovine serum albumin protein exhibiting relatively high binding constant values.

Mechanical properties of tetra-PEG gels with supercoiled network structure

We investigate the effects of swelling and deswelling on the mechanical properties of tetra-polyethylene glycol gels with the precisely tuned polymerization degree of network strand (Nc) and polymer volume fraction at preparation (ϕ0) by varying the fraction of interest (ϕm). The ϕm-dependence of the elastic modulus exhibits a crossover at ϕc due to large contraction of the network strands (supercoiling) accompanying deswelling. The Obukhov model successfully describes the ϕm-dependence of the elastic modulus. We estimate the fractal dimension of network strands (Df) by analyzing the stress-elongation relationships at high stretching using Pincus blob concept. In the supercoiling region, Df increases with an increase in ϕm, which suggests that the gyration radius of network strands decreases with deswelling in affine manner. The extensibility increases with an increase in ϕm because the deswelling reduces the distance between the neighboring junctions. These findings will help to understand the structure and formation mechanism of supercoiling.

Mechanical Strain Generated by RNA Polymerase during Transcription Initiation can Drive Structural Changes in DNA Topology that Relieve Repression

DNA in cells is often topologically closed, and in some cases, tightly looped by proteins typically associated with transcriptional repression. During transcription initiation, RNA polymerase (RNAP) is challenged to open duplex DNA, wherein RNAP generates torque that consequently overtwists DNA flanking the melted promoter. As initiation proceeds, the transcription bubble expands as RNAP synthesizes downstream RNA while maintaining upstream DNA contacts. We hypothesized that the mechanical strain imparted to DNA during initiation would repress transcription from DNA templates that restrict the relief of torsional stress. To test our hypothesis, we constructed circular DNA templates that are 100 to 108 bp in size (i.e., each initially twisted to various degrees) and quantified transcription initiation by the bacteriophage T7 RNAP. We find that transcriptional repression during initiation is dependent on the sign and magnitude of initial twist within the DNA templates. Surprisingly, however, we observe that for the most overtwisted templates, repression is relieved at positions beyond the promoter that are dependent on the initial DNA twist. To interpret these results, we used elastic rod and molecular dynamics simulations to predict the structures of both the RNAP and the circular DNA template during initiation. Our modeling studies confirm that RNAP is capable of overtwisting the circular DNA template to the point of buckling from a planar into a supercoiled conformation. Further analysis reveals that initial DNA twist determines at what position along the template supercoiling will occur during initiation and that adopting a supercoiled structure substantially relieves the torque encountered by the RNAP. Our results demonstrate that repression of RNAP during initiation is determined not only by the initial mechanics of the DNA template, but also by the torque generated by RNAP itself.

Self-Assembled Superhelical Structure of Poly(N-vinylcarbazole)-Based Donor–Acceptor Polymer at the Air–Water Interface

Preparation of helical polymers designed for mimicking biological helices and developing supramolecular helical assemblies with a controlled helix sense still remains a major challenge. A previously unknown superhelical structure constructed by achiral random copolymer poly(N-vinylcarbazole) (PVK) with pendant azobenzene chromophores and terminal nitro moieties (PVK–AZO–NO2) at the air/water interface was systematically investigated by CD spectroscopy, AFM, and TEM. The compression of the interfacial films may induce the helices to coil up and form large superhelices with the opposite handedness in a manner analogous to forming the supercoiled tertiary structure of DNA. The structure model for the self-assembly of PVK–AZO–NO2 into suprahelical structures at the air/water interface was also proposed.

Oxidative stress induces mitochondrial DNA damage and cytotoxicity through independent mechanisms in human cancer cells.

Intrinsic oxidative stress through increased production of reactive oxygen species (ROS) is associated with carcinogenic transformation, cell toxicity, and DNA damage. Mitochondrial DNA (mtDNA) is a natural surrogate to oxidative DNA damage. MtDNA damage results in the loss of its supercoiled structure and is readily detectable using a novel, supercoiling-sensitive real-time PCR method. Our studies have demonstrated that mtDNA damage, as measured by DNA strand breaks and copy number depletion, is very sensitive to exogenous H2O2 but independent of endogenous ROS production in both prostate cancer and normal cells. In contrast, aggressive prostate cancer cells exhibit a more than 10-fold sensitivity to H2O2-induced cell toxicity than normal cells, and a cascade of secondary ROS production is a critical determinant to the differential response. We propose a new paradigm to account for different mechanisms governing cellular oxidative stress, cell toxicity, and DNA damage with important ramifications in devising new techniques and strategies in prostate cancer prevention and treatment.

RETRACTED ARTICLE: Drying of a plasmid containing formulation: chitosan as a protecting agent

BackgroundThe purpose of the study. Along with research on development of more efficient gene delivery systems, it is necessary to search on stabilization processes to extend their active life span. Chitosan is a nontoxic, biocompatible and available gene delivery carrier. The aim of this study was to assess the ability of this polymer to preserve transfection efficiency during spray-drying and a modified freeze-drying process in the presence of commonly used excipients.MethodMolecular weight of chitosan was reduced by a chemical reaction and achieved low molecular weight chitosan (LMWC) was complexed with pDNA. Obtained nanocomplex suspensions were diluted by solutions of lactose and leucine, and these formulations were spray dried or freeze dried using a modified technique. Size, polydispersity index, zeta potential, intensity of supercoiled DNA band on gel electrophoresis, and transfection efficiency of reconstituted nanocomplexes were compared with freshly prepared ones.Results and major conclusionSize distribution profiles of both freeze dried, and 13 out of 16 spray-dried nanocomplexes remained identical to freshly prepared ones. LMWC protected up to 100% of supercoiled structure of pDNA in both processes, although DNA degradation was higher in spray-drying of the nanocomplexes prepared with low N/P ratios. Both techniques preserved transfection efficiency similarly even in lower N/P ratios, where supercoiled DNA content of spray dried formulations was lower than freeze-dried ones. Leucine did not show a significant effect on properties of the processed nanocomplexes. It can be concluded that LMWC can protect DNA structure and transfection efficiency in both processes even in the presence of leucine.

Drying of a plasmid containing formulation: chitosan as a protecting agent

BackgroundAlong with research on development of more efficient gene delivery systems, it is necessary to search on stabilization processes to extend their active life span. Chitosan is a nontoxic, biocompatible and available gene delivery carrier. The aim of this study was to assess the ability of this polymer to preserve transfection efficiency during spray-drying and a modified freeze-drying process in the presence of commonly used excipients.MethodsMolecular weight of chitosan was reduced by a chemical reaction and achieved low molecular weight chitosan (LMWC) was complexed with pDNA. Obtained nanocomplex suspensions were diluted by solutions of lactose and leucine, and these formulations were spray dried or freeze dried using a modified technique. Size, polydispersity index, zeta potential, intensity of supercoiled DNA band on gel electrophoresis, and transfection efficiency of reconstituted nanocomplexes were compared with freshly prepared ones.Results and conclusionSize distribution profiles of both freeze dried, and 13 out of 16 spray-dried nanocomplexes remained identical to freshly prepared ones. LMWC protected up to 100% of supercoiled structure of pDNA in both processes, although DNA degradation was higher in spray-drying of the nanocomplexes prepared with low N/P ratios. Both techniques preserved transfection efficiency similarly even in lower N/P ratios, where supercoiled DNA content of spray dried formulations was lower than freeze-dried ones. Leucine did not show a significant effect on properties of the processed nanocomplexes. It can be concluded that LMWC can protect DNA structure and transfection efficiency in both processes even in the presence of leucine.

Investigation of a predicted N-terminal amphipathic α-helix using atomistic molecular dynamics simulation of a complete prototype poliovirus virion

The wild type 1 poliovirus capsid was first described in atomic detail in 1985 using X-ray crystallography. Numerous poliovirus capsid structures have been produced since, but none resolved the spatial positioning and conformation of a predicted N-terminal α-helix of the capsid protein VP1, which is considered critical to virus replication. We studied the helical structure under varying conditions using in silico reconstruction and atomistic molecular dynamics (MD) simulation methods based on the available poliovirus capsid atom coordinate data. MD simulations were performed on the detached N-terminal VP1 helix, the biologically active pentamer form of the pre-virion structure, reconstructed empty virus capsids and a full virion containing the poliovirus RNA genome in the form of a supercoiled structure. The N-terminal α-helix structure proved to be stable and amphipathic under all conditions studied. We propose that a combination of spatial disorder and proximity to the genomic RNA made this particular structure difficult to resolve by X-ray crystallography. Given the similarity of our in silico model of poliovirus compared to X-ray crystallography data, we consider computational methods to be a useful complement to the study of picornaviruses and other viruses that exhibit icosahedral symmetry.

Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin-Based Engineered Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8 Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Structure and Dynamics of Supercoiled DNA in Confinement

Cells must package their chromosomes within a volume whose characteristic length is orders of magnitude smaller than the genomic length. When sufficiently twisted, DNA assumes a supercoiled form, aiding in this necessary compaction, as suggested in the case of Caulobacter crescentus. Given the crucial role supercoiling plays in bacterial DNA packaging, an elucidation of supercoiling structure and dynamics under confinement promises to shed light on critical living processes during replication as well as genome accessibility during transcription. We explore the structure and dynamics of supercoiled DNA by modeling the strand as a self-interacting, elastic beaded chain with twist and conduct Brownian Dynamics simulations under various geometries and degrees of confinement. The mean end-to-end distance between DNA segments exhibits a scaling of 0.25 with genome contour length, consistent with recent experiments in Caulobacter and suggesting a randomly branched supercoiled configuration. The scaling subsequently becomes linear at lengths comparable to the size of the enclosure. The mean-distance dynamic behavior at various length scales is highly sensitive to degree of confinement, and we identify the biologically relevant time scales of relaxation. Short genome lengths relax relatively fast with power-law scaling at all confinements while highly confined DNA is impeded from fully exploring its configurational space, resulting in delayed relaxation at large length scales. In this regime, the DNA segments become effectively quenched, resembling a stiff, glassy state that prohibits large-scale movement. We delineate the length-dependent transitions between these dynamic states and discuss their biological consequences in light of genome accessibility and reorganization.

Self-Assembled Peptide Architecture with a Tooth Shape: Folding into Shape

Molecular self-assembly is the spontaneous association of molecules into structured aggregates by which nature builds complex functional systems. While numerous examples have focused on 2D self-assembly to understand the underlying mechanism and mimic this process to create artificial nano- and microstructures, limited progress has been made toward 3D self-assembly on the molecular level. Here we show that a helical β-peptide foldamer, an artificial protein fragment, with well-defined secondary structure self-assembles to form an unprecedented 3D molecular architecture with a molar tooth shape in a controlled manner in aqueous solution. Powder X-ray diffraction analysis, combined with global optimization and Rietveld refinement, allowed us to propose its molecular arrangement. We found that four individual left-handed helical monomers constitute a right-handed superhelix in a unit cell of the assembly, similar to that found in the supercoiled structure of collagen.

Long‐Term structural changes of plasmid DNA studied by atomic force microscopy

Long-term stability of plasmid DNA (pDNA) conformations is critical in many research areas, especially those concerning future gene therapy. Despite its importance, the time-evolution of pDNA structures has rarely been studied at a molecular resolution. Here, the time-evolution of pDNA solutions spanning four years was observed with atomic force microscopy (AFM). The AFM data show that the pDNA molecules changed over time from isolated supercoiled structures, to aggregated supercoiled structures, to thin, branched network structures, and finally to wider, branched network structures. Additional topographical analysis of the AFM data suggests that the actions of residual proteins could be the main mechanism for the structural changes in our laboratory-prepared pDNA.

Nucleation at the DNA supercoiling transition

Twisting DNA under a constant applied force reveals a thermally activated transition into a state with a supercoiled structure known as a plectoneme. Using transition-state theory, we predict the rate of this plectoneme nucleation to be of order 10(4) Hz. We reconcile this with experiments that have measured hopping rates of order 10 Hz by noting that the viscous drag on the bead used to manipulate the DNA limits the measured rate. We find that the intrinsic bending caused by disorder in the base-pair sequence is important for understanding the free-energy barrier that governs the transition. Both analytic and numerical methods are used in the calculations. We provide extensive details on the numerical methods for simulating the elastic rod model with and without disorder.

Potential Pitfalls In Quantitative Real-Time PCR for Molecular Monitoring of BCR-ABL In the Management of CML.

AbstractAbstract 1228Due to a lack of standardized methodologies and reporting, results of BCR-ABL transcripts by quantitative real-time PCR (qPCR) are difficult to evaluate and compare between laboratories in molecular monitoring of BCR-ABL in CML. Although progress has been made in harmonization of reporting BCR-ABL quantification, such as the introduction of the International Scale, the standardization of each step in qPCR for BCR-ABL quantification is important. The majority of laboratories use an absolute approach with a standard curve to quantitate the transcript level of BCR-ABL. A number of sources are available for standards, such as RNA, cDNA, and plasmid. Among those, plasmid DNA is the most widely used. Since the standard is the most important factor in the absolute approach, in this study different preparations of plasmid DNA were investigated by qPCR with a TaqMan probe to evaluate their amplification rate. BCR-ABL b3a2 and ABL were individually cloned into pGEM-T Easy vector, and each plasmid DNA was isolated with Qiagen Plasmid Maxi kit. After quantification, DNA was diluted to obtain E6 copy number as templates for qPCR. For data comparison, a common threshhold was set in the middle of the exponential phase and Ct values were obtained. A small Ct value indicates a high amplification rate. Multiple preparations of purified completely linearized plasmid DNA for both b3a2 and ABL have the Ct values of 17.90 ± 0.19 and 17.94 ± 0.44 respectively. These values were very similar or equal to the Cts of purified b3a2 and ABL DNA fragments derived from RT-PCR. However, the non-linearized circular plasmid DNA only had 20.36 ± 0.08 for b3a2 and 20.31 ± 0.13 for ABL when the same number of templates was analyzed in the same qPCR indicating a 5.5 fold deficiency in amplification rate. Although the amplification efficiency obtained from both linearized and circular plasmids were very similar in the range of 3.3±0.3, a deficiency in the amplification rate from the circular plasmid indicates that the complicated super coiled structure in the circular plasmid DNA may interfere with the amplification. After plasmid DNA was completely linearized with three individual restriction enzymes, aliquots of known amounts of plasmid DNA were adjusted to E6 copy number and analyzed without further purification. Depending on the restriction enzyme the amplification rate of non-purified plasmid varied from 69 to almost 100% compared with purified linearized plasmid DNA. To mimic the purification procedure, Rnase A was added to the restriction digestion mixture to remove RNA contamination. Without purification, the addition of RNase A significantly inhibits the amplification rate. Furthermore, standards from Ipsogen were compared in the study. From multiple concentration points in comparison of the standards, Ipsogen standards had a Ct value of 1.43 more than the purified completely linearized plasmid DNA with 2.7 fold deficiency in amplification rate. Since the Ct value of the sample is the same, the standards with low amplification rate would over-estimate the copy number of the sample. In theory, the copy ratio of the fusion gene (b3a2) to the control gene (ABL) should be the same, independent on the amplification rate of the standards. However, in practical experiments with a high amplification rate of the standards, the copy ratio is more accurate and reproducible when the samples have a low copy number of BCR-ABL. The combination of incomplete linearization of plasmid DNA in restriction enzyme digestion, non-purification of the linearized plasmid and contamination of interfering substances may together decrease the amplification rate. The preparation of purified completely linearized plasmid provides a standard way to obtain standards for qPCR in molecular monitoring of the BCR-ABL transcript level in the management of CML patients as well as for qPCR in other usages. Disclosures:No relevant conflicts of interest to declare.

The healing of full-thickness burns treated by using plasmid DNA encoding VEGF-165 activated collagen–chitosan dermal equivalents

Repair of deep burn by use of the dermal equivalent relies strongly on the angiogenesis and thereby the regeneration of dermis. To enhance the dermal regeneration, in this study plasmid DNA encoding vascular endothelial growth factor-165 (VEGF-165)/N,N,N-trimethyl chitosan chloride (TMC) complexes were loaded into a bilayer porous collagen–chitosan/silicone membrane dermal equivalents (BDEs), which were applied for treatment of full-thickness burn wounds. The DNA released from the collagen–chitosan scaffold could remain its supercoiled structure but its degree was decayed along with the prolongation of incubation time. The released DNA could transfect HEK293 cells in vitro with decayed efficiency too. Human umbilical vein endothelial cells (HUVECs) in vitro cultured in the scaffold loaded with TMC/pDNA-VEGF complexes expressed a significantly higher level of VEGF and showed higher viability than those cultured in the controls, i.e. blank scaffold, and scaffolds loaded with naked pDNA-VEGF and TMC/pDNA-eGFP, respectively. The four different BDEs were then transplanted in porcine full-thickness burn wounds. Results showed that the TMC/pDNA-VEGF group had a significantly higher number of newly-formed and mature blood vessels, and fastest regeneration of the dermis. RT-qPCR and western blotting found that the experimental group also had the highest expression of VEGF, CD31 and α-SMA in both mRNA and protein levels. Furthermore, ultra-thin skin grafting was performed on the regenerated dermis 14 days later, leading to complete repair of the burn wounds with normal histology. Moreover, the tensile strength of the repaired tissue increased along with the time prolongation of post grafting, resulting in a value of approximately 70% of the normal skin at 105 days.

Quantized Folding of Plasmid DNA Condensed with Block Catiomer into Characteristic Rod Structures Promoting Transgene Efficacy

Highly regulated folding of plasmid DNA (pDNA) through polyion complexation with the synthetic block catiomer, poly(ethylene glycol)-block-poly(L-lysine) (PEG-PLys), was found to occur in such a way that rod structures are formed with a quantized length of 1/2(n + 1) of the original pDNA length folding by n times. The folding process of pDNA was elucidated with regard to rigidity of the double-stranded DNA structure and topological restriction of the supercoiled closed-circular form, and a mechanism based on Euler's buckling theory was proposed. Folded pDNA exhibited higher gene expression efficiency compared to naked pDNA in a cell-free transcription/translation assay system, indicating that the packaging of pDNA into a polyion complex core surrounded by a PEG palisade is a promising strategy for constructing nonviral gene carrier systems. Extension of this finding may provide a reasonable model to further understand the packaging mechanism of supercoiled DNA structures in nature.

Preparation of DNA films for studies under vacuum conditions

Experiments were carried out to determine the optimum conditions required for the preparation of uniform films of supercoiled plasmid DNA to be used in irradiation experiments under high vacuum conditions. Investigations reveal that significant damage to the DNA molecules occurs due to the evacuation process when films were formed from DNA samples in ultra high purity water only. A variety of bases were tested for their possible protective capabilities and sodium hydroxide solution was found to be the most effective in maintaining the supercoiled structure of plasmid DNA during the preparation process. Using a transmission electron microscope we also examined the structure of the DNA films which are formed upon evacuation and how the proposed adducts influence the preparation process. It was found that the addition of bases cause the DNA to aggregate, noting that a base is required for the stability of the DNA molecules. The experimental results presented in this paper show that it may not be possible to perform experiments on so-called pure DNA under vacuum with no stabilizers being added to the sample before the evacuation process.

Gene delivery using dimethyldidodecylammonium bromide-coated PLGA nanoparticles

In this present work we describe a poly(lactic- co-glycolic acid) (PLGA) nanoparticle formulation for intracellular delivery of plasmid DNA. This formulation was developed to encapsulate DNA within PLGA nanoparticles that combined salting out and emulsion–evaporation processes. This process reduced the requirement for sonication which can induce degradation of the DNA. A monodispersed nanoparticle population with a mean diameter of approximately 240 nm was produced, entrapping a model plasmid DNA in both supercoiled and open circular structures. To induce endosomal escape of the nanoparticles, a superficial cationic charge was introduced using positively charged surfactants cetyl trimethylammonium bromide (CTAB) and dimethyldidodecylammonium bromide (DMAB), which resulted in elevated zeta potentials. As expected, both cationic coatings reduced cell viability, but at equivalent positive zeta potentials, the DMAB coated nanoparticles induced significantly less cytotoxicity than those coated with CTAB. Fluorescence and transmission electron microscopy demonstrated that the DMAB coated cationic nanoparticles were able to evade the endosomal lumen and localise in the cytosol of treated cells. Consequently, DMAB coated PLGA nanoparticles loaded with a GFP reporter plasmid exhibited significant improvements in transfection efficiencies with comparison to non-modified particles, highlighting their functional usefulness. These nanoparticles may be useful in delivery of gene therapies to targeted cells.