Sustained expression in mammalian cells with DNA complexed with chitosan nanoparticles

This work investigates the preparation and in vitro efficiency of chitosan gene transfection systems. Chitosan was used to prepare nanoparticles with a size range of 40-200 nm as determined using photon correlation spectroscopy (PCS) and 40-80 nm as determined using transmission electron microscopy (TEM). The ability of particles to complex DNA was investigated using gel retardation. Plasmid DNA pGL3-Control encoding firefly luciferase and pCH110 encoding beta-galactosidase were used as reporter genes. For transfection 293 human embryonal kidney cells and Chinese hamster ovary (CHO-K1) cells were used. The expression of luciferase was assayed and expressed as relative light units per milligram of protein (RLU/mg protein). Results showed that these chitosan particles have potential as vectors for the transfer of DNA into mammalian cells. Cellular transfection by the chitosan-pGL3-Control particles showed a sustained expression of the luciferase gene for about 10 days. Commercial transfection reagents, SuperFect and Lipofectin were also used. In contrast to chitosan particles, the duration of expression for both SuperFect and Lipofectin was only about 2 days. Agarose gel electrophoresis and displacement experiments using polyaspartic acid indicated a probable multiple interaction between DNA and chitosan whilst the interaction between DNA and the polyamidoamine dendrimer appears to be only ionic interaction. No toxic effect on the mammalian cells was seen with chitosan. SuperFect and Lipofectin however, were observed to engender marked cytotoxicity. Poly-D,L-lactide (PLA) nanoparticles (40-80 nm) and poly-L-lactide (PLLA) lamellae (2-6 microm) were also used to load DNA by an adsorption procedure, but these failed to give good expression data.     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys³³, Leu⁶⁷)₁₀₀-Orn₂₀, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 Å, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimentional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

DNA topology in a chromatin model system

The synthetic copolypeptide (Lys33, Leu67)100-Orn20, modeled on some general features of the histone sequences, has been found to supercoil the DNA double helix, wrapping it into a micelle, as a result of cohesive interactions between the polypeptide hydrophobic moieties. X-ray low-angle diffraction of complexes between the polypeptide and DNA is characterized by maxima at 50, 32, and 23 A, reminiscent of the chromatin pattern. The existence of a nucleosome-like structure along the DNA is suggested by gel electrophoresis analysis of DNA fragments after micrococcal nuclease digestion, showing the presence of a fragment of about 100 basepairs (bp) long. Topological experiments on the complexes with supercoiled as well as relaxed circular DNA by two-dimensional gel electrophoresis show the presence of left-handed superhelical turns. The results are in agreement with an intrinsic propensity of B-DNA to writhe into left-handed supercoils.     

HMG-D complexed to a bulge DNA: an NMR model

An NMR model is presented for the structure of HMG-D, one of the DROSOPHILA: counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.     

Virtual screening application of a model of full-length HIV-1 integrase complexed with viral DNA

To address the absence of experimental data on the full-length structure of HIV-1 integrase and the way it binds to viral and human DNA, we had previously [Karki, R. G.; Tang, Y.; Burke, T. R., Jr.; Nicklaus, M. C. J. Comput. Aided Mol. Des.2004, 18, 739] constructed models of full-length HIV-1 integrase complexed with models of viral and human DNA. Here we describe the discovery of novel HIV-1 integrase strand transfer inhibitors based on one of these models. Virtual screening methods including docking and filtering by predicted ADME/Tox properties yielded several microM level inhibitors of the strand transfer reaction catalyzed by wild-type HIV-1 integrase.     

Employment of cationic solid-lipid nanoparticles as RNA carriers

Gene transfer represents an important advance in the treatment of both genetic and acquired diseases. In this article, the suitability of cationically modified solid-lipid nanoparticles (SLN) as a nonviral vector for gene delivery was investigated, in order to obtain stable materials able to condense RNA. Cationic SLN were produced by microemulsion using Compritol ATO 888 as matrix lipid, Pluronic F68 as tenside, and dimethyldioctadecylammonium bromide (DDAB) as cationic lipid. The resulting particles were approximately 100 nm in size and showed a highly positive surface charge (+41 mV) in water. Size and shape were further characterized by scanning electron microscopy (SEM) measurements. Moreover, we utilized the sea urchin as a model system to test their applicability on a living organism. To evaluate cationic SLN ability to complex the in vitro transcribed Paracentrotus lividus bep3 RNA, we utilized both light scattering and gel mobility experiments, and protection by nuclease degradation was also investigated. By microinjection experiment, we demonstrated that the nanoparticles do not inference with the viability of the P. lividus embryo and the complex nanoparticles-bep3 permits movement of the RNA during its localization in the egg, suggesting that it could be a suitable system for gene delivery. Taken together, all these results indicate that the cationic SNL are a good RNA carrier for gene transfer system and the sea urchin a simple and versatile candidate to test biological properties of nanotechnology devices.     

Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.     

Silicon nanoparticles as a luminescent label to DNA

We successfully conjugated 1-2 nm diameter silicon nanoparticles to a 5'-amino-modified oligonucleotide (60mer) that contains a C6 linker between amide and phosphate groups. The conjugation was implemented via two photoinduced reactions followed by a DNA labeling step through formation of a carboxamide bond. Photoluminescence of the conjugates is dominated by two blue bands (400 and 450 nm maximal) under 340 nm excitation. The quantum yield of oligonucleotide-conjugated nanoparticles was determined to be 0.08 as measured against quinine sulfate in 0.1 M HClO(4) as a reference standard. We report a conjugation process that allows labeling of Si nanoparticles to an oligonucleotide in aqueous solutions. Ways to further optimize the procedure in order to achieve narrower and brighter photoluminescence are discussed.     

Detection of DNA using cationic polyhedral oligomeric silsesquioxane nanoparticles as the probe by resonance light scattering technique

A novel cationic polyhedral oligomeric silsesquioxane nanoparticle (cationic POSS) was synthesized and successfully used as a new probe for the detection of DNA by resonance light scattering technique (RLS). It was found that the electrostatic interaction of cationic POSS and DNA could obviously enhance the RLS signal, the enhanced RLS intensity at 360 nm was proportional to the concentration of nucleic acids within the range of 0.35-42.82 microg ml-1 for calf thymus DNA, the determination limit (3sigma) was 0.32 ng ml-1. The results showed this method was very sensitive, convenient, rapid and reproducible.     

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.     

Characterisation of three novel cationic lipids as liposomal complexes with DNA

Cationic lipids (CLs) are being increasingly exploited as transfection vectors for the delivery of DNA into eukaryotic cells. To obtain further insight to the complex formation and interactions between cationic liposomes and DNA, we characterised three novel cationic lipids, viz. bis[2-(11-phenoxyundecanoate)ethyl]-dimethylammonium bromide, N-hexadecyl-N-?10-[O-(4-acetoxy)-phenylundecanoate]ethyl?- dimethylammonium bromide, and bis[2-(11-butyloxyundecanoate)ethyl]dimethylammonium bromide. These lipids bear the same charged headgroup yet have different hydrophobic parts. Accordingly, we may anticipate their electrostatic interactions with DNA to be similar while differing in both thermal phase behaviour and physicochemical properties of their complexes with DNA. In keeping with the above all three lipids formed complexes with DNA as evidenced by light scattering, fluorescence spectroscopy and Langmuir film balance. Differential scanning calorimetry revealed very different phase behaviours for the binary mixtures of the three CLs with dimyristoylphosphatidylcholine and also provided evidence for DNA-induced lipid phase separation. These data were confirmed by compression isotherms and fluorescence microscopy of monolayers residing on an aqueous buffer, recorded both in the presence and absence of DNA. Importantly, binding to cationic liposomes appears to prevent thermal denaturation of DNA upon heating of the complexes. Likewise, renaturation of heat-treated DNA complexed with the cationic liposomes appears to be abolished as well.     

Transcription of chromatin from mouse fibroblasts

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended «light and a compact «heavy component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II).Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.     

Radiation breakage of DNA: a model based on random-walk chromatin structure

Monte Carlo computer software, called DNAbreak, has recently been developed to analyze observed non-random clustering of DNA double strand breaks in chromatin after exposure to densely ionizing radiation. The software models coarse-grained configurations of chromatin and radiation tracks, small-scale details being suppressed in order to obtain statistical results for larger scales, up to the size of a whole chromosome. We here give an analytic counterpart of the numerical model, useful for benchmarks, for elucidating the numerical results, for analyzing the assumptions of a more general but less mechanistic “randomly-located-clusters” formalism, and, potentially, for speeding up the calculations. The equations characterize multi-track DNA fragment-size distributions in terms of one-track action; an important step in extrapolating high-dose laboratory results to the much lower doses of main interest in environmental or occupational risk estimation. The approach can utilize the experimental information on DNA fragment-size distributions to draw inferences about large-scale chromatin geometry during cell-cycle interphase. Received: 2 March 2000 / Revised version: 2 February 2001 / Published online: 21 August 2001