10 research outputs found

    Reconnaissance moleculaire d'acides nucleiques : conception de regulateurs artificiels de l'expression genetique

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    SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

    Theories equationnelles et de contraintes pour la demonstration automatique en logique multi-modale

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    SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 82205 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

    Inhibiteurs bisubstrats de méthyltransférases virales

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    International audienceIn the last two decades, emerging viruses such as flaviviruses or coronaviruses have caused significant health andeconomic damage. Released upon infection in the host, methyltransferases are involved in the methylation of theviral messenger RNA cap. These methylations, on the N7 position of the guanosine and 2’ O position of the firstnucleotide transcribed in the cap, increase the stability of the viral messenger RNA in the cell while protecting itfrom the cellular receptors of the innate immune system. Their role is therefore essential and a deactivation of thesemethyltransferases could lead to the elimination of the virus by stimulation of the innate immune system. Followingthis objective, the work reported in this article concerns the design and evaluation of viral methyltransferaseinhibitors. The bisubstrate strategy was used to improve the specificity of these novel compoundsAu cours des deux dernières décennies, les virus émergents comme les Flavivirus ou les Coronavirus ont causé d'importants dommages sanitaires et économiques. Le complexe de réplication / transcription virale contient des enzymes essentielles au développement du virus. D'autres enzymes comme les méthyltransférases sont impliquées dans la méthylation de la coiffe de l'ARN messager viral. Ces méthylations, sur les positions N7 de la guanosine et 2'O du premier nucléotide transcrit dans la coiffe, augmentent la stabilité de l'ARN messager viral dans la cellule tout en le protégeant face aux récepteurs cellulaires du système immunitaire inné, comme RIG-I, qui ont la capacité de reconnaitre et entrainer l'élimination de cellules infectées. Leur rôle est donc primordial et une désactivation de ces méthyltransférases pourrait entrainer l'élimination du virus par stimulation du système immunitaire inné. Suivant cet objectif, les travaux de thèse reportés dans cet article concernent la synthèse et l'évaluation d'inhibiteurs de méthyltransférases virales. La conception de ces inhibiteurs repose sur une stratégie bisubstrat afin d'augmenter l'affinité et la spécificité de ces composés originaux

    FTIR and UV Spectroscopy Studies of Triplex Formation Between α-Oligonucleotides with Non-Ionic Phoshoramidate Linkages and DNA Targets

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    International audienceThe triplexes formed by pyrimidine alpha-oligodeoxynucleotides, 15mers alpha dT(15) or 12mers alpha dCT having dimethoxyethyl (PNHdiME), morpholino (PMOR) or propyl (PNHPr) non-ionic phosphoramidate linkages with DNA duplex targets have been investigated by UV and FTIR spectroscopy. Due to the decrease in the electrostatic repulsion between partner strands of identical lengths all modifications result in triplexes more stable than those formed with unmodified phosphodiester beta-oligodeoxynucleotides (beta-ODNs). Among the alpha-ODN third strands having C and T bases and non-ionic phosphoramidate linkages (alpha dCTPN) the most efficient modification is (PNHdiME). The enhanced third strand stability of the alpha dCTPN obtained as diastereoisomeric mixtures is attenuated by the steric hindrance of the PMOR linkages or by the hydrophobicity of the PNHPr linkages. All alpha dCTPN strands form triplexes even at neutral pH. In the most favorable case (PNHdiME), we show by FTIR spectroscopy that the triplex formed at pH 7 is held by Hoogsteen T*A.T triplets and in addition by an hydrogen bond between O6 of G and C of the third strand (Tm = 30 degrees C). The detection of protonated cytosines is correlated at pH 6 with a high stabilization of the triplex (Tm = 65 degrees C). While unfavorable steric effects are overcome with alpha anomers, the limitation of the pH dependence is not completely suppressed. Different triplexes are evidenced for non pH dependent phosphoramidate alpha-thymidilate strands (alpha dT(15)PN) interacting with a target duplex of identical length. At low ionic strength and DNA concentration we observe the binding to beta dA(15) either of alpha dT(15)PN as duplex strand and beta dT(15) as third strand, or of two hydrophobic alpha dT(15)PNHPr strands. An increase in the DNA and counterion concentration stabilizes the anionic target duplex and then the alpha dT(15)PN binds as Hoogsteen third strand

    The biolabile 2'-O-pivaloyloxymethyl modification in an RNA helix: an NMR solution structure

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    International audienceThe pivaloylloxymethyl (PivOM) group is a biolabile 2'-O-ribose protection that is under development in a prodrug-based approach for siRNA applications. Besides an expected cellular uptake, nucleic acid sequences carrying PivOM showed also increased nuclease resistance and, in most cases, an affinity for complementary RNA. The r(CGCU*ACGC)dT:r(GCGUAGCG)dT model duplex containing a single modified residue (U*) was synthesized and its solution structure was determined by NMR. The duplex showed a maintained A-RNA helix. In U*, both 2'-O-acetal ester side chain and ring pucker presented a notable rigid conformation. The PivOM moiety was oriented with the carbonyl group turned outside the minor groove and with trans,-ac and -ac torsion angles around the C2'-O2',O2'-CA and CA-OB1 bonds respectively. Gauche effects and dipolar interactions between the PivOM and the backbone appeared to be the predominant factors influencing the PivOM conformation and the orientation of the two supplementary H acceptors suggested that hydration could also play a role in the duplex stability

    FTIR and UV Spectroscopy Studies of Triplex Formation Between Pyrimidine Methoxyethylphosphoramidates α- Oligodeoxynucleotides and ds DNA Targets

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    International audienceThe ability of non-ionic methoxyethylphosphoramidate (PNHME) alpha-oligodeoxynucleotides (ODNs), alpha dT(15) and alpha dCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME alpha-ODNs formed much more stable triplexes than phosphodiester (PO) beta-ODNs did (Delta Tm = + 20 degrees C for alpha dCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by alpha dT(15) PNHME compared to phosphodiester ODNs with beta or alpha anomeric configuration. While beta dT(15) PO failed to form a triplex with a long beta dA(n) x beta dT(n) duplex, the Tm of the Hoogsteen part of the triplex formed by alpha dT(15) PNHME reached 40 degrees C. Moreover alpha dT(15) PNHME displaced the beta dT(15) strand of a shorter beta dA(15) x beta dT(15) duplex. The alpha dCT PNHME and alpha dT(15) PNHME third strands were found antiparallel in contrast to alpha dT(15) PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides alpha dT and alpha dC combining both replacements might contribute to the improve stability of the triplexes

    Solid-phase synthesis of 5′-triphosphate 2′-5′-oligoadenylates analogs with 3′-O-biolabile groups and their evaluation as RNase L activators and antiviral drugs.

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    International audience5′-Triphosphate 2′-5′-oligoadenylate (2-5A) is the central player in the 2-5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2-5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2-5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2-5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2-5A drug in an intact form offer an alternative approach to therapeutic use of 2-5A. Here we have synthesized the trimeric and tetrameric 2-5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3′-positions and a triphosphate at the 5′-end. Both analogs were able to activate RNase L and the production of the trimer 2-5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity

    Ecological catalysis and phytoextraction: symbiosis for future

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    International audienceMetallophyte plants derived from phytoextraction are used as starting materials to prepare novel polymetallic catalysts. Polymetallic catalyst activity is used in many Lewis acid catalyzed reactions according to the polymetallic catalyst preparation. The synergetic catalysis of these systems leads to efficient syntheses of complex biomolecules such as dihydropyrimidinone, 5'-capped DNA and RNA, and glycosyl aminoacid. These new polymetallic catalysts also bring new possibilities in Green Catalysis, that we named "Ecological Catalysis"

    X-ray structure and activities of an essential Mononegavirales L-protein domain

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    International audienceThe L protein of mononegaviruses harbours all catalytic activities for genome replication and transcription. It contains six conserved domains (CR-I to -VI; Fig. 1a). CR-III has been linked to polymerase and polyadenylation activity, CR-V to mRNA capping and CR-VI to cap methylation. However, how these activities are choreographed is poorly understood. Here we present the 2.2-Å X-ray structure and activities of CR-VI+, a portion of human Metapneumovirus L consisting of CR-VI and the poorly conserved region at its C terminus, the +domain. The CR-VI domain has a methyltransferase fold, which besides the typical S-adenosylmethionine-binding site (SAMP) also contains a novel pocket (NSP) that can accommodate a nucleoside. CR-VI lacks an obvious cap-binding site, and the SAMP-adjoining site holding the nucleotides undergoing methylation (SUBP) is unusually narrow because of the overhanging +domain. CR-VI+ sequentially methylates caps at their 2′O and N7 positions, and also displays nucleotide triphosphatase activity
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