Aptamer Structures A Preview into Regulatory Pathways?

AbstractThe crystal structure of a streptomycin binding RNA aptamer displays a novel bipartite fold able to clamp the antibiotic. In view of the recent findings that metabolites directly control mRNA translation, we might expect that similar structures exist in natural RNAs

Stabilities of HIV-1 DIS type RNA loop–loop interactions in vitro and in vivo

RNA loop–loop interactions are a prevalent motif in the formation of tertiary structure and are well suited to trigger molecular recognition between RNA molecules. We determined the stabilities of several loop–loop interactions with a constant 6 bp core sequence and varying unpaired flanking nucleotides and found that the flanking bases have a strong influence on the stability and ion dependence of the kissing complex. In general, the stabilities determined in 1 M Na(+) are equivalent to those in the presence of near physiological Mg(2+) concentrations. Therefore we further tested whether the stabilities determined in vitro and within yeast cells correlate, using a recently developed yeast RNA-hybrid system. For the majority of the loop types analyzed here, the melting temperatures determined in vitro are in good agreement with the relative β-galactosidase activity in yeast cells, showing that data derived from in vitro measurements reflect in vivo properties. The most stable interactions are the naturally occurring HIV-1 DIS MAL and LAI derived loops with the motif (5′ A(A)/(G)N(6)A 3′), emphasizing the crucial role of stable kissing complexes in HIV genome dimerization

Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells

Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues

Genetic Diversity, Morphological Uniformity and Polyketide Production in Dinoflagellates (Amphidinium, Dinoflagellata)

Dinoflagellates are an intriguing group of eukaryotes, showing many unusual morphological and genetic features. Some groups of dinoflagellates are morphologically highly uniform, despite indications of genetic diversity. The species Amphidinium carterae is abundant and cosmopolitan in marine environments, grows easily in culture, and has therefore been used as a ‘model’ dinoflagellate in research into dinoflagellate genetics, polyketide production and photosynthesis. We have investigated the diversity of ‘cryptic’ species of Amphidinium that are morphologically similar to A. carterae, including the very similar species Amphidinium massartii, based on light and electron microscopy, two nuclear gene regions (LSU rDNA and ITS rDNA) and one mitochondrial gene region (cytochrome b). We found that six genetically distinct cryptic species (clades) exist within the species A. massartii and four within A. carterae, and that these clades differ from one another in molecular sequences at levels comparable to other dinoflagellate species, genera or even families. Using primers based on an alignment of alveolate ketosynthase sequences, we isolated partial ketosynthase genes from several Amphidinium species. We compared these genes to known dinoflagellate ketosynthase genes and investigated the evolution and diversity of the strains of Amphidinium that produce them

SELECTION IN VITRO DE RIBOZYMES ALLOSTERIQUES POUR LE CONTROLE DE L'EXPRESSION DES GENES

NOUS CHERCHONS A DEVELOPPER UN SYSTEME DE CONTROLE DE L'EXPRESSION GENIQUE AYANT LES PROPRIETES SUIVANTES : PAS DE CO-EXPRESSION DE FACTEURS PROTEIQUES DE CONTROLE, USAGE D'UNE MOLECULE REGULATRICE NON TOXIQUE ET ACTIVATION DE L'EXPRESSION EN PRESENCE DE LA DROGUE. LA SELECTION IN VITRO EST UN OUTIL PERMETTANT L'ISOLATION D'ACIDES NUCLEIQUES EXTREMEMENT RARES. NOUS AVONS IMAGINE TIRER PARTI DE CETTE METHODE POUR ISOLER DES ARN CATALYTIQUES (RIBOZYMES) REGULABLES PAR UNE DROGUE ARBITRAIREMENT CHOISIE (DOXYCYCLINE OU PEFLOXACINE). INSERES DANS UN ARN MESSAGER (ARNM), UN TEL RIBOZYME DEVRAIT INHIBIBER L'EXPRESSION DU GENE EN SE CLIVANT, PROVOQUANT LA DEGRADATION DU TRANSCRIT. L'ADMINISTRATION DE LA MOLECULE REGULATRICE DEVRAIT EMPECHER LE CLIVAGE ET PERMETTRE UNE RESTAURATION DE L'EXPRESSION. NOUS AVONS INTRODUIT DANS LA SEQUENCE DU RIBOZYME A TETE DE MARTEAU UN SEGMENT ALEATOIRE, ET SOUMIS LA BIBLIOTHEQUE AINSI FORMEE A UNE SELECTION FONCTIONNELLE. APRES 16 CYCLES DE SELECTION DE PLUS EN PLUS STRINGENTE, NOUS AVONS PU OBSERVER DES CLONES REPONDANT A DES QUANTITES TRES FAIBLES DE LA DROGUE REGULATRICE (20 NM). DE PLUS CERTAINS CLONES SONT TRES SELECTIFS POUR LEUR LIGAND. DANS CERTAINS CAS, ILS SONT 10 000 FOIS PLUS SENSIBLES POUR LA DOXYCYCLINE QUE POUR LA TETRACYCLINE BIEN QUE CES DEUX MOLECULES NE DIFFERENT QUE PAR LA POSITION D'UN GROUPE HYDROXYLE. L'INTRODUCTION D'UNE MUTATION PONCTUELLE PERMET LA CONVERSION DES RIBOZYMES EN APTAMERES CLASSIQUES. DANS UN CAS, LA DETERMINATION D'UN MOTIF DE LIAISON MINIMUM A PERMIS L'ELABORATION D'HYPOTHESES QUANT AU MECANISME D'INHIBITION. PAR CONTRE, L'INTRODUCTION DES RIBOZYMES SELECTIONNES DANS UN ARNM N'A PAS PERMIS D'OBSERVER L'EFFET VOULU. POUR UN CLONE, LA DROGUE A INDUIT UNE INHIBITION DE L'EXPRESSION DU GENE RAPPORTEUR D'UN FACTEUR 2, CE QUI CORRESPOND A L'EFFET INVERSE A L'EFFET ATTENDU. CE RESULTAT LAISSE ENVISAGER QUE LA CAUSE N'EST PAS PERDUE ET SUGGESTE DES EXPERIENCES VISANT A L'ISOLEMENT DE SEQUENCES AYANT L'ACTIVITE APPROPRIEE. CE TRAVAIL A PERMIS LA DECOUVERTE DE RIBOZYMES ALLOSTERIQUES PLUS DE MILLE FOIS PLUS SENSIBLE A LA MOLECULE REGULATRICE PAR RAPPORT AUX RESULTATS PUBLIES ANTERIEUREMENT. DE PLUS, LA POSSIBILITE DE CONVERTIR EN APTAMERES LES CLONES SELECTIONNES INVITE A IMAGINER UN ROLE PLUS LARGE POUR CE TYPE DE SELECTION FONCTIONNELLE.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

A yeast RNA-hybrid system for the detection of RNA–RNA interactions in vivo

RNA–RNA interactions play a crucial role at many different levels of the cellular metabolism such as plasmid replication control, viral encapsidation, or transcriptional and translational regulation. Therefore, methods are necessary to investigate the molecular determinants of given interactions, including their stabilities, or to screen for new interacting partners. We designed an RNA-hybrid system in S. cerevisiae, based on the yeast three-hybrid system. In this setup, the activation of a reporter gene is dependent on the interaction of two RNAs. A loop–loop interaction similar to the dimerization initiation site of the HIV genome was used as a model system, demonstrating that in this novel RNA-hybrid system only cognate RNAs promote the activation of the reporter gene. Levels of reporter activation correlate well with interaction stabilities determined in vitro by UV melting analyses, suggesting that conditions used for the analysis of in vitro structural stabilities translate well into the intracellular environment. Furthermore, the system was applicable for a screen against a test library. Nine out of ten selected clones were identified as predicted interaction partners for the bait RNA. In summary, we present a yeast reporter system depending on RNA–RNA interactions, which can be used alternatively for analysis of known interactions or for screening libraries in search for new interaction partners

Vanishing GC-rich isochores in mammalian genomes.

To understand the origin and evolution of isochores-the peculiar spatial distribution of GC content within mammalian genomes-we analyzed the synonymous substitution pattern in coding sequences from closely related species in different mammalian orders. In primate and cetartiodactyls, GC-rich genes are undergoing a large excess of GC --> AT substitutions over AT --> GC substitutions: GC-rich isochores are slowly disappearing from the genome of these two mammalian orders. In rodents, our analyses suggest both a decrease in GC content of GC-rich isochores and an increase in GC-poor isochores, but more data will be necessary to assess the significance of this pattern. These observations question the conclusions of previous works that assumed that base composition was at equilibrium. Analysis of allele frequency in human polymorphism data, however, confirmed that in the GC-rich parts of the genome, GC alleles have a higher probability of fixation than AT alleles. This fixation bias appears not strong enough to overcome the large excess of GC --> AT mutations. Thus, whatever the evolutionary force (neutral or selective) at the origin of GC-rich isochores, this force is no longer effective in mammals. We propose a model based on the biased gene conversion hypothesis that accounts for the origin of GC-rich isochores in the ancestral amniote genome and for their decline in present-day mammals

() β-Galactosidase assay of the different loop–loop interactions investigated

<p><b>Copyright information:</b></p><p>Taken from "Stabilities of HIV-1 DIS type RNA loop–loop interactions and "</p><p>Nucleic Acids Research 2006;34(1):334-342.</p><p>Published online 12 Jan 2006</p><p>PMCID:PMC1331993.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> β-Galactosidase activity is expressed in Miller Units and was normalized relative to the activity of the XYLAI interaction. All hairpins were tested together with their cognate interaction partner (closed bars). As control all RNAs X and Y were assayed against non-cognate interaction partners, namely YΘ for RNAs X (light gray bars) and XΘ for RNAs Y (open bars). Furthermore transformants expressing only either XLAI-MS2 or m26-YLAI or no hairpin RNA at all are shown. s for the cognate kissing complexes at a concentration of 2 × 10 M are indicated. Error bars show the SD of two independent triplicate experiments. () Correlation between the and the β-galactosidase activity determined for kissing interactions containing 9 nt in the loop. (XYAA and YX0 contain 8 and 6 nt, respectively, and are not included.) () Schematic representation of cognate and non-cognate interactions considering XYLAI as example

Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology