Ribozymes, a new therapeutic strategy?

Las ribozimas son moléculas de RNA con actividad catalítica, capaces de catalizar reacciones químicas en el interior celular. En su contexto natural la gran mayoría de las ribozimas descritas llevan a cabo el procesamiento de otras moléculas de RNA. A diferencia de las ribonucleasas proteicas, las ribozimas presentan una gran especificidad de substrato, esta es la característica en la que se fundamenta la idea de que las ribozimas puedan ser utilizadas como supresores génicos específicos y servir de base para el desarrollo de nuevos agentes terapéuticos. La necesidad de disponer de nuevas estrategias terapéuticas se hace más evidente cuando surgen enfermedades contra las cuales los fármacos y terapias empleados no son eficientes. La repercusión social de determinadas enfermedades de difícil o escasa probabilidad de curación (SIDA, Cáncer, etc.), ha agudizado la necesidad de buscar tratamientos alternativos. La actuación directa sobre la información genética responsable de la dolencia, es una de las aproximaciones experimentales en desarrollo. En este sentido, la utilización de ácidos nucleicos como agentes supresores constituye, desde hace algunos años, una línea de investigación importante. Como resultado están surgiendo un conjunto de nuevas tecnologías en tomo a conceptos tales como oligonucleótidos antisense, ribozimas y triples hélices. Las ribozimas actuarían bloqueando la transmisión de información genética a nivel del RNA mediante la destrucción de genomas RNA (virus RNA) o de mRNAs. El diseño y la aplicación efectiva de ribozimas con nuevas especificidades requiere un profundo conocimiento acerca de estas moléculas así como de sus mecanismos de reconocimiento e interacción con los RNAs substrato. En este trabajo se describen los RNAs catalíticos mejor caracterizados, así como las diferentes estrategias desarrolladas hasta el momento para la utilización de las ribozimas como inactivadores génicos.Ribozymes are RNA molecules endowed with catalytic activity, they are able to cataIyze chemical reactions inside the cells. In their natural environment, most of the described ribozymes carry out processing of other RNA molecule. In contrast to the protein ribonucleases, ribozymes exhibit a high-substrate specificity. This feature is the base for the potential use of ribozymes as specific gene suppressors and therefore to develop new therapeutic agents. The need of new therapeutic strategies is even more significant with the rising of diseases against which the existent drugs and therapies do not show the desirable efficiency. The social repercussions of some diseases of difficult or low probability of control (AIDS, Cancer, etc.), has made more acute the need of looking for altemative treatments. Among the experimental approaches that are being carried out is the direct action on the genetic information responsible of such diseases. On this line, several strategies have emerged in the past few years, mainly based on the use of nucleic acids as specific gene suppressors, e.g. antisense oligonucleotides, ribozymes and triplex helix. The ribozymes would act blocking the transmission of genetic information at the RNA level, mediating destruction of RNA genomes (RNA virus) or mRNAs. A deep understanding of the mechanisms used by these molecules for the recognition and interaction with their RNA substrates as well as of these molecules themselves is necessary for the design and effective application of ribozymes with new specificity. In this work we describe the best characterized catalytic RNAs, as well as the different strategies developed for their use as gene tic suppressors

RNA self-cleavage activated by ultraviolet light-induced oxidation

A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5′-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C 79 and CSFV A45 in some molecules and at the immediately adjacent 5′-positions HCV U 78 and CSFV U 44 in others. Among other reaction products, the majority of biochemically active products detected contained 3′-phosphate and 5′-phosphate-end groups at the newly generated termini, along with a much lower amount of 3′-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (∼33nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures. © 2012 The Author(s).http://dx.doi.org/10.1093/nar/gkr82

Hairpin ribozyme-antisense RNA constructs can act as molecular lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5′- and 3′-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-α (TNF-α) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns

Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences

The tobamovirus resistance gene L3 of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinenseL3 gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L3-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L3 gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L3 locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus

Modulating RNA structure and catalysis: lessons from small cleaving ribozymes

RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today’s knowledge in the field

Ribozimas, ¿una nueva estrategia terapéutica?

Las ribozimas son moléculas de RNA con actividad catalítica, capaces de catalizar reacciones químicas en el interior celular. En su contexto natural la gran mayoría de las ribozimas descritas llevan a cabo el procesamiento de otras moléculas de RNA. A diferencia de las ribonucleasas proteicas, las ribozimas presentan una gran especificidad de substrato, esta es la característica en la que se fundamenta la idea de que las ribozimas puedan ser utilizadas como supresores génicos específicos y servir de base para el desarrollo de nuevos agentes terapéuticos. La necesidad de disponer de nuevas estrategias terapéuticas se hace más evidente cuando surgen enfermedades contra las cuales los fármacos y terapias empleados no son eficientes. La repercusión social de determinadas enfermedades de difícil o escasa probabilidad de curación (SIDA, Cáncer, etc.), ha agudizado la necesidad de buscar tratamientos alternativos. La actuación directa sobre la información genética responsable de la dolencia, es una de las aproximaciones experimentales en desarrollo. En este sentido, la utilización de ácidos nucleicos como agentes supresores constituye, desde hace algunos años, una línea de investigación importante. Como resultado están surgiendo un conjunto de nuevas tecnologías en tomo a conceptos tales como oligonucleótidos antisense, ribozimas y triples hélices. Las ribozimas actuarían bloqueando la transmisión de información genética a nivel del RNA mediante la destrucción de genomas RNA (virus RNA) o de mRNAs. El diseño y la aplicación efectiva de ribozimas con nuevas especificidades requiere un profundo conocimiento acerca de estas moléculas así como de sus mecanismos de reconocimiento e interacción con los RNAs substrato. En este trabajo se describen los RNAs catalíticos mejor caracterizados, así como las diferentes estrategias desarrolladas hasta el momento para la utilización de las ribozimas como inactivadores génicos.Ribozymes are RNA molecules endowed with catalytic activity, they are able to cataIyze chemical reactions inside the cells. In their natural environment, most of the described ribozymes carry out processing of other RNA molecule. In contrast to the protein ribonucleases, ribozymes exhibit a high-substrate specificity. This feature is the base for the potential use of ribozymes as specific gene suppressors and therefore to develop new therapeutic agents. The need of new therapeutic strategies is even more significant with the rising of diseases against which the existent drugs and therapies do not show the desirable efficiency. The social repercussions of some diseases of difficult or low probability of control (AIDS, Cancer, etc.), has made more acute the need of looking for altemative treatments. Among the experimental approaches that are being carried out is the direct action on the genetic information responsible of such diseases. On this line, several strategies have emerged in the past few years, mainly based on the use of nucleic acids as specific gene suppressors, e.g. antisense oligonucleotides, ribozymes and triplex helix. The ribozymes would act blocking the transmission of genetic information at the RNA level, mediating destruction of RNA genomes (RNA virus) or mRNAs. A deep understanding of the mechanisms used by these molecules for the recognition and interaction with their RNA substrates as well as of these molecules themselves is necessary for the design and effective application of ribozymes with new specificity. In this work we describe the best characterized catalytic RNAs, as well as the different strategies developed for their use as gene tic suppressors

Analysis of the Mechanism of Action of the Antisense RNA That Controls the Replication of the repABC Plasmid p42d ▿ †

Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5′ end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli