Change of the higher order structure of DNA induced by the complexation with intercalating synthetic polymer, as is visualized by fluorescence microscopy (original) (raw)
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Complexation of DNA with Cationic Polymers
Studia Universitatis Babeș-Bolyai Chemia, 2018
Polyethylenimine (PEI) represents the most extensively used non-viral vector for gene delivery. The complexation between nucleic acids and PEI chains is intimately related to electrostatic interactions of the positively charged amine groups with the negatively charged phosphate groups. All-atom molecular dynamics simulations of alternatively protonated PEI chains, DNA and, respectively, polyplexes thereof in solution were performed. Our results reveal an increase in gyration radius of solvated PEI chains in the presence of DNA. In order to understand the major changes in DNA properties, the impact of PEI chains on the ionic environment of DNA is described in detail. In addition, the amine-phosphate contact analysis provides valuable insight into the formation mechanism of PEI/DNA complexes.
Journal of Biotechnology, 2007
The purpose of the study was to investigate the influence of cationic polymer structure on the formation of DNA-polycation complexes and their transfection activity. Primary, tertiary, and quaternary polyamines with molecular masses ranging from 8000 to 200,000 were investigated. DNA-cationic polymer interaction was characterized by low gradient viscometry, dynamic light scattering, circular dichroism, UV spectrometry, flow birefringence, DNA electrophoresis, and electron microscopy. Transfection activity of the complexes was evaluated by the expression of reporter gene (-galactosidase) and using synthetic FITC-labelled oligonucleotides. Complex formation was found to be dependent on the structure and molecular weight of the polymer and the ionic strength of the solution. Secondary DNA structure in complexes was not disrupted, and DNA was protected from protonation. Cell lines of different origin were used for testing of transfection activity of the complexes. The sensitivity of the cells to transfection was established to be highly dependent on the cell line. DNA-polycation complexes are non-toxic according to MTT. Polyallylamine, and polydimethylaminoethylmethacrylate were found to be the most promising polycations for gene delivery. Transfection efficacy of their complexes with DNA to T-98G cells reaches up to 90-100%. It was found that optimal molecular mass of polydimethylaminoethylmethacrylate is in the range of 8000-50,000 Da.
Colloids and Surfaces B-biointerfaces, 2008
The aggregation of the cationic polymer-plasmid DNA complexes of two commonly used polymers, polyethyleneimine (PEI) and poly-l-lysine (PLL) were systematically compared. The complexation was studied in 5% glucose solution at 25 • C using dynamic light scattering and isothermal titration calorimetry. The aggregation of the complexes was controlled by addition of the surfactant polyoxyethylene stearate (POES). The stability of the complexes was evaluated using dextran sulphate (DS) as relaxing agent. The relaxation of the complexes in the presence of DS was studied using agarose gel electrophoresis. This study elucidates the role of surfactant in controlling the size of the PEI/pDNA complex and reveals the differences of the two polymers as complexing agents.
Biomacromolecules, 2004
Intermolecular complexes of genomic polydisperse DNA with synthetic polycations have been studied. Two cationic polymers have been used, a homopolymer poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its analogue grafted with poly(oxyethylene). The amount of poly(oxyethylene) grafts in the copolymer was 15 mol % and M w of the graft was 200 g/mol. Salmon DNA (sodium salt) was used. The average molecular weight (M w ) of DNA was 10.4 × 10 6 g/mol. Conductivity, pH, and dynamic light scattering studies were used to characterize the complexes. The size and shape of the polyelectrolyte complex particles have been studied as a function of the cation-to-anion ratio in aqueous solutions of varying ionic strengths. The polyelectrolyte complexes have extremely narrow size distributions taking into account the polydispersity of the polyelectrolytes studied. The poly(oxyethylene) grafts on PMOTAC promote the formation of small colloidally stabile complex particles. Addition of salt shifts the macroscopic phase separation toward lower polycation content; that is, complexes partly phase separate with the mixing ratios far from 1:1. Further addition of salt to the turbid, partly phase separated solution results in the dissociation of complexes and the polycation and DNA dissolve as individual chains.
Colloids and Surfaces B: Biointerfaces, 2008
We synthesized a cationic polymer, poly(PEGMA)-4N, which has brush-like chains and four positively charged amino groups at the end of the molecules. DNA condensation induced by poly(PEGMA)-4N was investigated through electrophoresis assay by its ability to retard DNA mobility and to inhibit HindIII enzyme cleavage. The detailed structures of DNA condensates induced by poly(PEGMA)-4N were observed through atomic force microscopy (AFM). Interactions between polymers and DNA are mainly attributed into depletion effect and electrostatic interaction. Positively charged amino groups in poly(PEGMA)-4N interact with DNA through electrostatic interaction, and depletion effect also takes effect because poly(PEGMA)-4N is a flexible polymer. Comparing the contributions that the two interactions gave in DNA condensation process, we found that depletion effect played a major role compared with electrostatic interaction.
Biomaterials, 2005
A series of comb-type copolymers comprised of various polycation backbones and dextran (Dex) side chains were prepared to study the DNA/copolymer interaction. While the cationic copolymers with a lower degree of dextran grafts maintained an ability to condense DNA molecules into a globule form those with a higher degree of dextran grafting interacted with DNA without inducing DNA condensation. The structural differences in cationic backbones diversely influenced DNA hybridization as evaluated by circular dichroism (CD) spectrometry and UV-melting analyses. The copolymer having a polyallylamine (PAA) backbone induced B-A-type transformation of DNA duplex, whereas the copolymers having either a-poly(l-lysine) (aPLL) or e-poly(l-lysine) (ePLL) backbone induced B-C-type transformation. The PAA copolymer is the first example of the artificial polymer that induces B-A-type transformation under physiologically relevant condition. UV-melting analyses of DNA strands indicated that the aPLL copolymers showed the highest stabilization efficacy toward poly(dA) Á poly(dT) duplex and poly(dA) Á 2poly(dT) triplex without affecting reversibility of inter DNA association. Melting temperatures (T m ) of the triplex increased from 38 C to 99 C by the addition of the aPLL copolymer with an appropriate grafting degree. While the PAA copolymers had higher density of cationic groups along the backbone than aPLL copolymers, these copolymers moderately increased T m of the DNA triplex. The PAA copolymer caused considerable hysteresis in thermal melting/reassociation processes. Note that the ePLL copolymers increased T m of the DNA triplex and not the duplex, suggesting their potential as a triplex selective stabilizer. Chemical structures of the cationic backbones of the copolymers were characteristically affected on the copolymer/DNA interaction even if their backbones were surrounded by abundant side chains (> 65 wt%) of dextran. The study suggested that tailor-made design of ''functional polycounterion'' is a strategy to engineer molecular assembling of DNA. r
Recognition of DNA Topology in Reactions between Plasmid DNA and Cationic Copolymers
Journal of the American Chemical Society, 2000
This study for the first time demonstrates phenomenon of recognition of DNA tertiary structure by the synthetic polycationic molecules. Effects of DNA topology were evaluated using supercoiled and linearized forms of plasmid DNA (scDNA and lDNA). Recognition is achieved by using relatively simple chemical structures interacting with the DNA. Two polycations modified with water-soluble poly(ethylene glycol) (PEG) chains, PEG-block-poly(N-methyl-4-vinylpyridinium sulfate) (PEG-b-PVP) and PEG-graft-polyethyleneimine (PEG-g-PEI) were used. When added to the mixtures of lDNA and scDNA, PEO-b-PVP selectively bound to scDNA, while lDNA remained free. In contrast, PEO-g-PEI interacted with both forms of the DNA present in the mixture. Distinct behavior of two copolymers was attributed to the differences in their structure, particularly, charge density of the polycation blocks. Relatively small variation in the polycation ionization state can result in drastic changes in its behavior upon interaction with DNA. Particularly, the change of pH from 7.0 to 5.0, increasing the charge density of PEI block in PEO-g-PEI, was also accompanied by the appearance of recognition phenomena. These findings uncover possibilities for the control of the processes of DNA incorporation in the complexes with cationic species by altering the DNA topology, which may have practical significance in using these complexes for gene delivery. Non-viral gene delivery systems based on DNA complexes with polycations have recently attracted significant attention. 1 These complexes form spontaneously as a result of cooperative electrostatic interactions between phosphate groups of the DNA and oppositely charged groups of the polycation. Characterization of the reactions of polyion coupling between DNA and polycation and interactions of formed polycation/DNA com
Journal of Biotechnology, 2006
Atomic force microscopy (AFM) is used to describe the formation process of polymer/DNA complexes. Two main objectives of this research are presented. The first one is to apply AFM as an effective tool to analyse DNA molecules and different polycation/DNA complexes in order to evaluate their degree of condensation (size and shape). The other one is to search for a relationship between the condensation state of DNA and its transfection efficiency. In this study, linear methacrylate based polymers and globular SuperFect polymers are used in order to induce DNA condensation. Ternary complexes, composed of methacrylate based polymers and polyethylene glycol (PEG)-based copolymers, are also investigated. AFM allows us to confirm good condensation conditions and relate them (or not) to transfection efficiencies. These AFM results (obtained after drying in air) are compared with measurements deduced from Dynamic Light Scattering (DLS) experiments performed in water. This comparison allowed us to identify the structural modifications resulting from deposition on the mica surface.
Secondary Structure of DNA Is Recognized by Slightly Cross-Linked Cationic Hydrogel
Journal of the American Chemical Society, 2002
Interaction of salmon sperm DNA (300-500 bp) and ultrahigh molecular mass DNA (166 kbp) from bacteriophage T4dC with linear poly(N-diallyl-N-dimethylammonium chloride) (PDADMAC) and slightly cross-linked (#) PDADMAC (#PDADMAC) hydrogel in water has been studied by means of UV-spectroscopy, ultracentrifugation, atomic force, and fluorescence microscopy (FM). It is found that the linear polycation induced compaction of either native (double-stranded) or denatured (single-stranded) DNA by forming PDADMAC-DNA interpolyelectrolyte complexes (IPEC)s. At the same time, #PDADMAC hydrogel is able to distinguish between native and denatured DNA. Native DNA is adsorbed and captured in the hydrogel surface layer, while denatured DNA diffuses to the hydrogel interior until the whole hydrogel sample is transformed into the cross-linked IPEC. Both native and denatured DNA can be completely released from the hydrogel in appropriate conditions with no degradation by adding a low molecular salt. The data observed using conventional physicochemical methods with respect to DNA of a moderate molecular mass remarkably correlate with the pictures directly observed for ultrahigh molecular mass DNA in dynamics by using FM.