Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.
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ABSTRACT:
SUBMITTER: Hendrix C
PROVIDER: S-EPMC1217097 | biostudies-other | 1996 Mar
REPOSITORIES: biostudies-other
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Project description:Hammerhead ribozyme is the smallest and best characterized catalytic RNA-cleaving ribozyme. It has been reported as potential therapeutic tools to manipulate the expression of target genes. However, most of naturally occurring hammerhead ribozymes process self-cleavage rather than cleave substrate RNA in trans, and its high intracellular activity relies on the tertiary interaction of Loop II and steam I bulge, resulting in decreased performance as applied in gene silencing. We described a direct intracellular selection method to evolve hammerhead variants based on trans-cleavage mode via using a toxin gene as the reporter. And a dual fluorescence proteins system has also been established to quantitatively evaluate the efficiency of selected ribozymes in the cell. Based on this selection strategy, we obtained three mutants with enhanced intracellular cleaving activity compared to wide type hammerhead ribozyme. The best one, TX-2 was revealed to possess better and consistent gene knockdown ability at different positions on diverse targeted mRNA either in prokaryotic or eukaryotic cells than wild-type hammerhead ribozyme. These observations imply the efficiency of the intracellular selection method of the trans-acting ribozyme and the potentials of improved ribozyme variants for research and therapeutic purposes.
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Project description:We obtained a partial sequence of mouse calretinin mRNA from cDNA clones, and designed hammerhead ribozymes to cleave positions within it. With a view to optimising hammerhead ribozymes for eliminating the mRNA in vivo, we varied the length and sequence of the three duplex 'arms' and measured the cleavage of long RNA substrates in vitro at 37 degrees C (as well as 50 degrees C). Precise cleavage occurred, but it could only go to completion with a large excess of ribozyme. The evidence suggests that the rate-limiting step with a large target is not the cleavage, but the formation of the active ribozyme: substrate complex. The efficiency varied unpredictably according to the target site, the length of the substrate RNA, and the length of the ribozyme; secondary structure in vitro may be responsible. We particularly investigated the degree of sequence-specificity. Some mismatches could be tolerated, but shortening of the total basepairing with the substrate to less than 14 bp drastically reduced activity, implying that interaction with weakly-matched RNAs is unlikely to be a serious problem in vivo. These results suggest that specific and complete cleavage of a mRNA in vivo should be possible, given high-level expression of a ribozyme against a favourable target site.
| S-EPMC310633 | biostudies-other
Project description:Natural hammerhead ribozymes are mostly found in some viroid and viroid-like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis-acting hammerheads self-cleave much faster than trans-acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100-fold reduction of the self-cleavage constant, whereas engineering a trans-acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo.
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Project description:Because oxygen and selenium are in the same group (Family VI) in the periodic table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on the structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucleoside 5'-(alpha-P-seleno)triphosphates (NTPalphaSe, including A, C, G, and U). We found that T7 RNA polymerase recognizes NTPalphaSe Sp diastereomers as well as the natural NTPs, whereas NTPalphaSe Rp diastereomers are neither substrates nor inhibitors. We also demonstrated the catalytic activity of these Se-derivatized hammerhead ribozymes by cleaving the RNA substrate, and we found that these phosphoroselenoate ribozymes can be as active as the native one. These hammerhead ribozymes site-specifically mutagenized by selenium reveal the close relationship between the catalytic activities and the replaced oxygen atoms, which provides insight on the participation of oxygen in catalysis or intramolecular interaction. This demonstrates a convenient strategy for the mechanistic study of functional RNAs. In addition, the active ribozymes site-specifically derivatized by selenium will allow for convenient MAD phasing in X-ray crystal structure studies.
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Project description:We report here that a single additional trans-Hoogsteen base-pairing interaction in the minimal hammerhead ribozyme transforms an RNA sequence possessing typically modest catalytic activity into one possessing greatly enhanced catalytic activity that is instead typical of full-length natural hammerhead RNAs that have additional extensive tertiary contact interactions. Formation of this additional base-pairing interaction requires only that the substrate RNA sequence contains a U at a position seven nucleotides 3' to the cleavage site. No additions or changes are required in the minimal hammerhead ribozyme enzyme strand sequence (providing that the naturally occurring GUGA tetraloop of Stem II is maintained). This finding unambiguously demonstrates that a single Hoogsteen base-pairing interaction, in full-length hammerheads possessing this interaction, is sufficient for stabilizing the ribozyme active site, including alignment of the attacking nucleophile for the required inline hammerhead ribozyme reaction mechanism. This finding also implies that the idiosyncratic arrays of additional tertiary contacts observed in all naturally occurring full-length hammerhead sequences have evolved to prevent deleterious alternative pairing interactions within the context of the variety of natural sequences arising in vivo. Finally, this finding greatly simplifies and rationalizes the design of fast-cleaving engineered synthetic ribozymes as RNA nucleolytic reagents and as subjects for enzyme kinetics and mechanistic investigations.
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Project description:Using a computer program designed to search for RNA structural motifs in sequence databases, we have found a hammerhead ribozyme domain encoded in the Smalpha repetitive DNA of Schistosoma mansoni. Transcripts of these repeats are expressed as long multimeric precursor RNAs that cleave in vitro and in vivo into unit-length fragments. This RNA domain is able to engage in both cis and trans cleavage typical of the hammerhead ribozyme. Further computer analysis of S. mansoni DNA identified a potential trans cleavage site in the gene coding for a synaptobrevin-like protein, and RNA transcribed from this gene was efficiently cleaved by the Smalpha ribozyme in vitro. Similar families of repeats containing the hammerhead domain were found in the closely related Schistosoma haematobium and Schistosomatium douthitti species but were not present in Schistosoma japonicum or Heterobilharzia americana, suggesting that the hammerhead domain was not acquired from a common schistosome ancestor.
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Project description:Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/.
| S-EPMC4191386 | biostudies-literature
Project description:Deep sequencing of viral or bacterial nucleic acids monitors the presence and diversity of microbes in select populations and locations. Metagenomic study of mammalian viromes can help trace paths of viral transmissions within or between species. High throughput sequencing of patient and untreated sewage microbiomes showed many sequences with no similarity to genomic sequences of known function or origin. To estimate the distribution of functional RNAs in these microbiomes, we used the hammerhead ribozyme (HHR) motif to search for sequences capable of assuming its three-way junction fold. Although only two of the three possible natural HHR topologies had been known, our analysis revealed highly active ribozymes that terminated in any of the three stems. The most abundant of these are type II HHRs, one of which is the fastest natural cis-acting HHR yet discovered. Altogether, 13 ribozymes were confirmed in vitro, but only one showed sequence similarity to previously described HHRs. Sequences surrounding the ribozymes do not generally show similarity to known genes, except in one case, where a ribozyme is immediately preceded by a bacterial RadC gene. We demonstrate that a structure-based search for a known functional RNA is a powerful tool for analysis of metagenomic datasets, complementing sequence alignments.
| S-EPMC3048661 | biostudies-literature
Project description:Small ribozymes have been regarded as living fossils of a prebiotic RNA world that would have remained in the genomes of modern organisms. In this study, we report the ultraconserved occurrence of hammerhead ribozymes in Amniota genomes (reptiles, birds and mammals, including humans), similar to those described previously in amphibians and platyhelminth parasites. The ribozymes mapped to intronic regions of different genes, such as the tumour suppressor RECK in birds and mammals, a mammalian tumour antigen and the dystrobrevin beta in lizards and birds. In vitro characterization confirmed a high self-cleavage activity, whereas analysis of RECK-expressed sequence tags revealed fusion events between the in vivo self-cleaved intron and U5 or U6 small nuclear RNA fragments. Together, these results suggest a conserved role for these ribozymes in messenger RNA biogenesis.
| S-EPMC2933863 | biostudies-other
Project description:Ribozymes are small catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and site-specific cleavage. Trans-cleaving ribozymes can inhibit translation of genes at the messenger RNA (mRNA) level in both eukaryotic and prokaryotic systems and are thus useful tools for studies of gene function. However, identification of target sites for efficient cleavage poses a challenge. Here, we have considered a number of structural and thermodynamic parameters that can affect the efficiency of target cleavage, in an attempt to identify rules for the selection of functional ribozymes.We employed the Sfold program for RNA secondary structure prediction, to account for the likely population of target structures that co-exist in dynamic equilibrium for a specific mRNA molecule. We designed and prepared 15 hammerhead ribozymes to target GUC cleavage sites in the mRNA of the breast cancer resistance protein (BCRP). These ribozymes were tested, and their catalytic activities were measured in vitro. We found that target disruption energy owing to the alteration of the local target structure necessary for ribozyme binding, and the total energy change of the ribozyme-target hybridization, are two significant parameters for prediction of ribozyme activity. Importantly, target disruption energy is the major contributor to the predictability of ribozyme activity by the total energy change. Furthermore, for a target-site specific ribozyme, incorrect folding of the catalytic core, or interactions involving the two binding arms and the end sequences of the catalytic core, can have detrimental effects on ribozyme activity.The findings from this study suggest rules for structure-based rational design of trans-cleaving hammerhead ribozymes in gene knockdown studies. Tools implementing these rules are available from the Sribo module and the Srna module of the Sfold program available through Web server at http://sfold.wadsworth.org.
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