Targeted inhibition of the hepatitis C internal ribosomal entry site genomic RNA with oligonucleotide conjugates

Hepatitis C is a major public health concern, with an estimated 170 million people infected worldwide and an urgent need for new drug development. An attractive therapeutic approach is to prevent the ‘cap-independent’ translation initiation of the viral proteins by interfering with both the structure and function of the hepatitis C viral internal ribosomal entry site (HCV IRES). Towards this goal, we report the design, synthesis and purification of novel bi-functional molecules containing DNA or RNA antisenses attached to functional groups performing RNA hydrolysis. These 5′ or 3′-coupled conjugates bind the HCV IRES with affinity and specificity and elicit targeted hydrolysis of the viral genomic RNA after short (1 h) incubation at low (500 nM) concentration at 37°C in vitro. Additional secondary cleavage sites are induced and their mapping within the RNA structure indicates that functional domains IIIb-e are excised from the IRES that, based on cryo-EM studies, becomes incapable of binding the small ribosomal subunit and initiation factor 3 (eIF3). All these molecules inhibit, in a dose-dependent manner, the ‘IRES-dependent’ translation in vitro. The 5′-coupled imidazole conjugate reduces viral protein synthesis by half at a 300 nM concentration (IC50), corresponding to a 4-fold increase of activity when compared to the naked oligonucleotide. These new conjugates are now being tested for activity on infected hepatic cell lines

Mechanisms of base selection by human single-stranded selective monofunctional uracil-DNA glycosylase

hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved

Activity of core modified 10-23 DNAzymes against HCV

The highly conserved untranslated regions of the hepatitis C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2′R)-, (2′S)-2′-deoxy-2′-C-methyl- and -2′-O-methylnucleosides at various positions of the catalytic core were assayed against the 5′-internal ribosome entry site element (5′-IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes′ cores were jointly modified with 2′-C-methyl- and 2′-O-methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48 h of transfection. Different regions of the HCV IRES were explored with unmodified 10–23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES-firefly luciferase translation-dependent RNA (IRES-FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh-7 human hepatocarcinoma cell line. Inhibition of IRES-dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.Fil: Robaldo, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Berzal Herranz, Alfredo. Instituto de Parasitología y Biomedicina “López-Neyra”; EspañaFil: Montserrat, Javier Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad Nacional de General Sarmiento. Instituto de Ciencias; ArgentinaFil: Iribarren, Adolfo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Area Química. Laboratorio de Biotransformaciones; Argentin

Etude de la réparation enzymatique de lésions de l'ADN en utilisant des oligonucléotides fixés sur support

GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Intérêt de la supplémentation en arginine dans la prévention cardiovasculaire (une revue des études cliniques)

PARIS-BIUP (751062107) / SudocSudocFranceF

Targeted degradation of the hepatitis C virus internal ribosome entry site genomic RNA with oligonucleotide conjugates.

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