12 research outputs found
Target-dependent on/off switch increases ribozyme fidelity
Ribozymes, RNA molecules that catalyze the cleavage of RNA substrates, provide an interesting alternative to the RNA interference (RNAi) approach to gene inactivation, especially given the fact that RNAi seems to trigger an immunological response. Unfortunately, the limited substrate specificity of ribozymes is considered to be a significant hurdle in their development as molecular tools. Here, we report the molecular engineering of a ribozyme possessing a new biosensor module that switches the cleavage activity from ‘off’ (a ‘safety lock’) to ‘on’ solely in the presence of the appropriate RNA target substrate. Both proof-of-concept and the mechanism of action of this man-made riboswitch are demonstrated using hepatitis delta virus ribozymes that cleave RNA transcripts derived from the hepatitis B and C viruses. To our knowledge, this is the first report of a ribozyme bearing a target-dependent module that is activated by its RNA substrate, an arrangement which greatly diminishes non-specific effects. This new approach provides a highly specific and improved tool with significant potential for application in the fields of both functional genomics and gene therapy
Silencing of Amyloid Precursor Protein Expression Using a New Engineered Delta Ribozyme
Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD
RiboSubstrates: a web application addressing the cleavage specificities of ribozymes in designated genomes
BACKGROUND: RNA-dependent gene silencing is becoming a routine tool used in laboratories worldwide. One of the important remaining hurdles in the selection of the target sequence, if not the most important one, is the designing of tools that have minimal off-target effects (i.e. cleaves only the desired sequence). Increasingly, in the current dawn of the post-genomic era, there is a heavy reliance on tools that are suitable for high-throughput functional genomics, consequently more and more bioinformatic software is becoming available. However, to date none have been designed to satisfy the ever-increasing need for the accurate selection of targets for a specific silencing reagent. RESULTS: In order to overcome this hurdle we have developed RiboSubstrates . This integrated bioinformatic software permits the searching of a cDNA database for all potential substrates for a given ribozyme. This includes the mRNAs that perfectly match the specific requirements of a given ribozyme, as well those including Wobble base pairs and mismatches. The results generated allow rapid selection of sequences suitable as targets for RNA degradation. The current web-based RiboSubstrates version permits the identification of potential gene targets for both SOFA-HDV ribozymes and for hammerhead ribozymes. Moreover, a minimal template for the search of siRNAs is also available. This flexible and reliable tool is easily adaptable for use with any RNA tool (i.e. other ribozymes, deoxyribozymes and antisense), and may use the information present in any cDNA bank. CONCLUSION: RiboSubstrates should become an essential step for all, even including "non-RNA biologists", who endeavor to develop a gene-inactivation system
A guanine-linked end-effect is a sensitive reporter of charge flow through DNA and RNA double helices
Développement d'un système d'inactivation de gènes basé sur le ribozyme delta
L'information générée par le décodage du génome humain nous permet de contempler davantage toute la complexité de la cellule et de ses processus biochimiques. Plus de 25 000 gènes encodant des protéines y ont été répertoriés. De plus, du point de vue épissage alternatif, une modification post-transcriptionnelle, les possibilités de protéines résultantes de l'expression de ces gènes sont presque infinies. La plupart de ces gènes n'ont pas encore de fonction leur ayant été associée. Les composés organiques chimiques employés tous les jours par des millions de personnes pour un mal de tête ou bien des maux d'estomac, ne viendront pas à bout de l'identification fonctionnelle de ces gènes. Souvent, ces composés agissent au niveau protéique, soit sur une batterie de protéines et ainsi en découle un manque de spécificité. Il est alors impératif de développer de nouveaux outils thérapeutiques très spécifiques avec un champ d'action bien précis, soit le ciblage du produit des gènes. Mon projet de recherche s'inscrit exactement dans ce type de réalisation. Nous avons développé un système d'inactivation de gènes basé sur le ribozyme delta, un enzyme à ARN avec une activité catalytique. Premièrement, nous avons élaboré des stratégies pour sélectionner des sites actifs sur le substrat ARN permettant une activité forte du ribozyme, malgré un environnement souvent non propice à cette coupure. Ces méthodes ont été comparées et permettent aujourd'hui un choix et une sélection beaucoup plus rapide de cibles potentielles. Par la suite, nous avons appliqué ce ribozyme delta dans le but d'inactiver l'expression d'un gène, une convertase, exprimée de façon endogène et ce, dans une culture de cellules murines. Il en résulte l'inhibition presque complète de l'expression du gène, tant au niveau de l'ARN que de la protéine. Nous avons démontré que l'activité de cette convertase n'est pas compensée par d'autres protéines de la même famille. Ces résultats ont aussi permis d'amener l'hypothèse de la présence d'un nouveau substrat pour cette convertase. Par conséquent, cette étude démontre sans ambiguité l'efficacité et la spécificité du ribozyme delta en milieu cellulaire. Cependant, puisque la recherche nous pousse toujours à dépasser les barrières de l'inconnu, ce que nous connaissons de mieux n'est toujours pas assez. Dans le but d'augmenter la spécificité du ribozyme delta, nous avons, par design rationnel, créé un nouveau module nommé SOFA, pour"Specific On/ofF Adapter", permettant un gain accru d'activité et de spécificité.--Résumé abrégé par UMI
Functional characterization of the SOFA delta ribozyme
Molecular engineering has led to the development of a novel target-dependent riboswitch that increases δribozyme fidelity. This δ ribozyme possesses a specific on/off adapter (SOFA) that switches the cleavage activity from off (a “safety lock”) to on solely in the presence of the desired RNA substrate. In this report, we investigate the influence of both the structure and the sequence of each domain of the SOFA module. Analysis of the cleavage activity, using a large collection of substrates and SOFA-ribozyme mutants, together with RNase H probing provided several insights into the nature of the sequence and the optimal design of each domain of the SOFA module. For example, we determined that (1) the optimal size of the blocker sequence, which keeps the ribozyme off in the absence of the substrate, is 4 nucleotides (nt); (2) a single nucleotide difference between the substrate and the biosensor domain, which is responsible for the initial binding of the substrate that subsequently switches the SOFA-ribozyme on, is sufficient to cause nonrecognition of the appropriate substrate; (3) the stabilizer, which joins the 5′ and 3′ ends of the SOFA-ribozyme, plays only a structural role; and (4) the optimal spacer sequence, which serves to separate the binding regions of the biosensor and catalytic domain of the ribozyme on the substrate, is from 1 to 5 nt long. Together, these data should facilitate the design of more efficient SOFA-ribozymes with significant potential for many applications in gene-inactivation systems
Short RNA duplexes guide sequence-dependent cleavage by human Dicer
Dicer is a member of the double-stranded (ds) RNA-specific ribonuclease III (RNase III) family that is required for RNA processing and degradation. Like most members of the RNase III family, Dicer possesses a dsRNA binding domain and cleaves long RNA duplexes in vitro. In this study, Dicer substrate selectivity was examined using bipartite substrates. These experiments revealed that an RNA helix possessing a 2-nucleotide (nt) 3′-overhang may bind and direct sequence-specific Dicer-mediated cleavage in trans at a fixed distance from the 3′-end overhang. Chemical modifications of the substrate indicate that the presence of the ribose 2′-hydroxyl group is not required for Dicer binding, but some located near the scissile bonds are needed for RNA cleavage. This suggests a flexible mechanism for substrate selectivity that recognizes the overall shape of an RNA helix. Examination of the structure of natural pre-microRNAs (pre-miRNAs) suggests that they may form bipartite substrates with complementary mRNA sequences, and thus induce seed-independent Dicer cleavage. Indeed, in vitro, natural pre-miRNA directed sequence-specific Dicer-mediated cleavage in trans by supporting the formation of a substrate mimic