Multimodal logic programming using equational and order-sorted logic

AbstractIn our previous works a method for automated theorem proving in modal logic, based on algebraic and equational techniques, was proposed. In this paper we extend the method to multimodal logic and apply it to modal logic programming. Multimodal systems under consideration have a finite number of pairs of modal operators (♢i, □i) of any type among KD, KT, KD4, KT4, KF, and interaction axioms of the form □iA → □jA. We define a translation from such logical systems to specially tailored equational theories of classical order-sorted logic, preserving satisfiability, and then use SLD E-resolution for theorem proving in these theories

mRNA maturation in giant viruses: variation on a theme

International audienceGiant viruses from the Mimiviridae family replicate entirely in their host cytoplasm where their genes are transcribed by a viral transcription apparatus. mRNA polyadenylation uniquely occurs at hairpin-forming palindromic sequences terminating viral transcripts. Here we show that a conserved gene cluster both encode the enzyme responsible for the hairpin cleavage and the viral polyA polymerases (vPAP). Unexpectedly, the vPAPs are homodimeric and uniquely self-processive. The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication. A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails. These findings elucidate how the arm wrestling between hosts and their viruses to access the translation machinery is taking place in Mimiviridae

Molecular Basis for Nucleotide Conservation at the Ends of the Dengue Virus Genome

International audienceThe dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5PolDV of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5PolDV contains a specific priming site for adenosine 59-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn 2+ is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5PolDV ensures the conservation of the 59-adenosine by strongly discriminating against viral templates containing an erroneous 39-end nucleotide in the presence of Mg 2+. In the presence of Mn2+, NS5Pol DV is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo

Incorporation de nucléosides modifiés dans des a-oligonucléosides N-alkylphosphoramidates et hybridation avec des cibles nucléiques simple et double brin

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Nouvelle méthode de synthèse d'ARN et préparation de prodrogues de siARN modifiés par des groupements acyloxyméthyle en position 2' des ribonucléosides

L'explosion des technologies basées sur l'ARN a relancé la recherche de méthodes plus efficaces pour sa synthèse chimique. De plus, l'utilisation des ARN en tant que médicament est fortement limitée par des problèmes de distribution et de stabilité dans l'organisme. Ces travaux se sont donc inscrits dans ces deux enjeux majeurs : améliorer la synthèse d'ARN naturels pour faire face à la forte demande de telles molécules et développer des ARN modifiés afin d'augmenter leur efficacité in vivo. Ce manuscrit rapporte une nouvelle méthode de synthèse chimique d'ARN basée sur l'utilisation de groupements acétalester pour la protection des hydroxyles en position 2' du ribose. Cette méthode a permis de simplifier l'accès à des séquences hétéropolymères d'ARN et est actuellement évaluée pour une application commerciale. Une deuxième partie présente la synthèse d'ARN modifiés portant, après élongation et déprotection, des groupements acétalester enzymolabiles en positions 2' du ribose. Les conditions d'obtention de ces ARN, désignés par pro-ARN en vue d'une application comme prodrogues de siARN dans le mécanisme de l'ARN interférence, ainsi que leurs propriétés sont décritesExpansion of RNA based technology has revived research for more powerful methodology for chemical RNA synthesis. Moreover, use of unmodified RNA as drug is restricted by poor cell permeation and low enzymatic stability in vivo. This work was related to these two challenges : find a new efficient strategy for chemical RNA synthesis and develop modified RNA in order to improve in vivo efficiency. Here, an original methodology for chemical RNA synthesis based on the protection of ribonucleosides 2'-hydroxyl by acetalester groups is presented. Thus, natural RNA heterosequences could be obtained with high purity and efficacy. This technology is currently under evaluation for commercial application. In a second part, we describe the synthesis of modified RNA bearing biolabile acetalester groups on nucleoside 2'-hydroxyl after full deprotection of other RNA functions. Synthesis and properties of this modified RNA, named pro-RNA in relation to their potential activity as siRNA prodrugs in RNA interference mechanism, are reportedMONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Synthèse chimique sur support solide d'ARN 5'-triphosphates et 5'-coiffés

Les ARN 5'-triphosphates (TP) et 5'-coiffés sont des molécules très convoitées des biologistes pour des études structurales par cristallographie ou comme outils thérapeutiques. Actuellement, ces oligonucléotides sont produits dans de faibles quantités via des procédés enzymatiques dépendants de la nature du nucléoside situé à l'extrémité 5'. Ces travaux se sont donc inscrits dans l'enjeu majeur de mettre au point une méthode chimique sur support solide qui permette l'accès à ces ARN 5'-fonctionnalisés en quantités importantes et sans restriction de séquences.Ce manuscrit rapporte une nouvelle méthode efficace de synthèse sur support solide d'ARN 5'-TP à grande échelle indépendante du nucléotide situé à l'extrémité 5' ou de la longueur de la séquence ARN. De plus, cette stratégie a été étendue avec quelques modifications à la synthèse d'analogues enzymatiquement plus stables de type: ARN 5'-b,g-méthylène-TP, -(a-P-thio)-TP et -(a-P-thio)-(b,g-méthylène)-TP.Une deuxième partie présente la synthèse supportée et l'évaluation antivirale de courts adénylates 2-5A 5'-TP portant des groupements enzymolabiles de type acétalesters en position 3' du ribose. Une dernière partie est consacrée à l'élaboration de nouvelles stratégies pour la synthèse d'ARN 5-coiffés. La première approche est un procédé en deux étapes où la structure coiffe (Gppp) est ajoutée sur support solide suivie d'une N7- méthylation enzymatique en solution, alors que la seconde est une méthode plus directe entièrement chimique. Par ailleurs, le couplage chimique de la coiffe sur les ARN supportés nécessite l'emploi d'un acide de Lewis. Ainsi l'utilisation de chlorures métalliques verts produits à partir de métaux de transition extraits de la biomasse a été évaluée dans la réaction du couplage de la coiffe sur les ARN.5'-triphosphates RNAand 5'-capped RNA are high valuable molecules which serve as important substrates for structural and mechanistic studies or drugs. To date, these oligonucléotides are produced by enzymatic process in low yields with a weak variability of the 5'-end. To overcome this bottleneck we aimed to develop a chemical access on solid-support of these 5'-functionalized RNA in great amount without any limitations in the RNA sequence.Here, a robust, reproducible, and scalable method for the solid-phase synthesis of 5 -triphosphate RNA is presented. Furthermore, this strategy was extended to produce enzymatic resistant analogs of the triphosphate counterpart like: 5'-b,g-methylene-TP, -(a-P-thio)-TP et -(a-P-thio)-(b,g-methylene)-TP RNA.In a second part, the solid-phase synthesis of short adenylates 2-5A 5'-TP bearing biolabile acetalester groups on nucleosides 3'-hydroxyls and their antiviral activities are described.Finally, we focused on developing two approaches for the production of 5'-capped RNA. One is a two-steps process which consists in the coupling of the cap structure on solid-support followed by an enzymatic N7-methylation in solution while the other one is a straightforward chemical strategy. Beside, the chemical coupling of the cap moiety on solid-supported RNA requires the use of Lewis acids. Then, the ability of green metal chlorides prepared from phytoextracted heavy metals to promote this reaction was studied.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Oligonucléotides d'anomérie [alpha] à liens internucléosidiques phosphoramidates cationiques

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

Oligonucleotides (ONs) have been envisaged for therapeutic applications for more than thirty years. However, their broad use requires overcoming several hurdles such as instability in biological fluids, low cell penetration, limited tissue distribution, and off-target effects. With this aim, many chemical modifications have been introduced into ONs definitively as a means of modifying and better improving their properties as gene silencing agents and some of them have been successful. Moreover, in the search for an alternative way to make efficient ON-based drugs, the general concept of prodrugs was applied to the oligonucleotide field. A prodrug is defined as a compound that undergoes transformations in vivo to yield the parent active drug under different stimuli. The interest in stimuli-responsive ONs for gene silencing functions has been notable in recent years. The ON prodrug strategies usually help to overcome limitations of natural ONs due to their low metabolic stability and poor delivery. Nevertheless, compared to permanent ON modifications, transient modifications in prodrugs offer the opportunity to regulate ON activity as a function of stimuli acting as switches. Generally, the ON prodrug is not active until it is triggered to release an unmodified ON. However, as it will be described in some examples, the opposite effect can be sought.This review examines ON modifications in response to various stimuli. These stimuli may be internal or external to the cell, chemical (glutathione), biochemical (enzymes), or physical (heat, light). For each stimulus, the discussion has been separated into sections corresponding to the site of the modification in the nucleotide: the internucleosidic phosphate, the nucleobase, the sugar or the extremities of ONs. Moreover, the review provides a current and detailed account of stimuli-responsive ONs with the main goal of gene silencing. However, for some stimuli-responsive ONs reported in this review, no application for controlling gene expression has been shown, but a certain potential in this field could be demonstrated. Additionally, other applications in different domains have been mentioned to extend the interest in such molecules