Production Of Dna Minicircles Less Than 250 Base Pairs Through A Novel Concentrated Dna Circularization Assay Enabling Minicircle Design With Nf-κb Inhibition Activity

Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmidfree method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide bluntended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2M. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids. © The Author(s) 2016.45

Adhesion to the digestive mucosa is not sufficient for durable persistence of different Lactobacillus fermentum strains in the digestive tract of mice

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Strain-controlled fluorescence polarization in a CdSe nanoplatelet–block copolymer composite

International audienceWe dispersed semi-conducting CdSe nanoplatelets within a styrene-butadiene-styrene block copolymer matrix. We could control the orientation of the nanoplatelets by stretching the resulting material, which provides a simple and reversible way of inducing fluorescence anisotropy. Such adjustable polarization effects are useful for modulating the optical response in composite materials. Hybrid materials made of anisotropic metallic or semi-conducting nanoparticles (NPs) dispersed in a polymer matrix have become an exciting class of nanocomposites with promising applications in electronics and optics. For example, the alignment of such materials can improve current transport in electronic devices or modulate the response in optical devices. 1, 2 However, two key points have to be addressed for this purpose. Firstly, a homogeneous dispersion of inorganic nanoparticles in polymeric templates is rather hard to achieve and most often requires surface modification. 3-7 Secondly, the macroscopic orientation of the material, which is required to exploit the anisotropic properties of the individual particles, must be controlled. To date, the orientation of the NPs in polymer matrices is mostly restricted to thin films. For example, nanoparticle alignment could be obtained by stretching a thermoplastic polymer at a temperature close to T g or by applying an electric field during solvent casting. 2, 8-10 In all cases, the orientation is irreversible, which precludes applications in opto-mechanical devices, for instance. We describe here how we achieved both a homogeneous dispersion of NPs in a thick film and the reversibility of the orientation in a composite material comprised of recently discovered CdSe nanoplatelets (NPLs) dispersed within a classical thermoplastic elastomer matrix. Indeed, CdSe NPLs have attracted much attention due to their outstanding spectroscopic properties. 11-16 These flat and square 2-dimensional photoluminescent nanoparticles display a sharp emission peak that can be precisely tuned by adjusting their thickness at the atomic level. Moreover, they have a very high quantum yield and fast recombination times of the carriers that are confined in one dimension. CdSe NPLs are generally synthesized in solution but they can also be deposited flat on a substrate 17 or aggregated edge-on in micronic needles. 18 To obtain a homogeneous composite material, we carefully adapted the BCP to the oleic acid ligand grafted on the NPLs. The polymer matrix that we have chosen is a commercial thermoplastic elastomer made of Styrene-Butadiene-Styrene (SBS) whose central block is of chemical nature and polarity close to those of oleic acid (Figure 1). A crucial advantage of such a matrix is the possibility to stretch it up to high strain levels in a reversible way at room temperature. We prepared homogeneous CdSe/SBS composite films with a CdSe volume fraction of 10% where both the SBS lamellar microstructure and the CdSe NPL spectroscopic properties are preserved. Figure 1. Components of the hybrid film. Left: schematic representation of a SBS copolymer chain. Center: schematic representation of a CdSe nanoplatelet covered with ligands. Right: photography of the hybrid film (Scale bar is 2 mm). The structural properties of these hybrid films were first studied by Transmission Electron Microscopy (TEM) (Figure 2a). The TEM images, which display light and dark grey bands corresponding to PS and PB domains respectively, clearly confirm the lamellar morphology of the SBS microstructure. Despite the high load in inorganic nanoparticles, the SBS microstructure in the hybrid films is therefore not much altered compared to that of the pure block copolymer. CdSe platelets have a strong electronic density but are very thin, which explains why the only particles clearly observed on the TEM images are those seen edge-on. Moreover, close inspection of the TEM images reveals that the CdSe platelets are mostly located in the PB domains, which was expected because of the chemical similarity of OA with PB. Very occasionally, a few platelets can be seen in the PS domains, possibly because of some ligand loss durin

Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity

Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmidfree method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide bluntended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2M. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids.45

Ultrastructural and immunocytochemical analysis of diploid germ cells isolated from fetal rabbit gonads

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Delipidating in vitro-produced bovine zygotes: effect on further development and consequences for freezability

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In vitro investigation of TiO2, Al2O3, Au nanoparticles and mutli-walled carbon nanotubes cyto- and genotoxicity on lung, kidney cells and hepatocytes

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Unité BIA-Jouy. Rapport préparé pour l'évaluation tenue les 3-4 avril 2002

323 ref. Annexes 71 p. *INRA Biométrie et Intelligence Artificielle Jouy-en-Josas (FRA) Diffusion du document : INRA Biométrie et Intelligence Artificielle Jouy-en-Josas (FRA)National audienc