Correction: Spiegelzymes® Mirror-Image Hammerhead Ribozymes and Mirror-Image DNAzymes, an Alternative to siRNAs and microRNAs to Cleave mRNAs In Vivo?
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SUBMITTER: The Staff
PROVIDER: S-EPMC3970976 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:With the discovery of small non-coding RNA (ncRNA) molecules as regulators for cellular processes, it became intriguing to develop technologies by which these regulators can be applied in molecular biology and molecular medicine. The application of ncRNAs has significantly increased our knowledge about the regulation and functions of a number of proteins in the cell. It is surprising that similar successes in applying these small ncRNAs in biotechnology and molecular medicine have so far been very limited. The reasons for these observations may lie in the high complexity in which these RNA regulators function in the cells and problems with their delivery, stability and specificity. Recently, we have described mirror-image hammerhead ribozymes and DNAzymes (Spiegelzymes®) which can sequence-specifically hydrolyse mirror-image nucleic acids, such as our mirror-image aptamers (Spiegelmers) discovered earlier. In this paper, we show for the first time that Spiegelzymes are capable of recognising complementary enantiomeric substrates (D-nucleic acids), and that they efficiently hydrolyse them at submillimolar magnesium concentrations and at physiologically relevant conditions. The Spiegelzymes are very stable in human sera, and do not require any protein factors for their function. They have the additional advantages of being non-toxic and non-immunogenic. The Spiegelzymes can be used for RNA silencing and also as therapeutic and diagnostic tools in medicine. We performed extensive three-dimensional molecular modelling experiments with mirror-image hammerhead ribozymes and DNAzymes interacting with D-RNA targets. We propose a model in which L/D-double helix structures can be formed by natural Watson-Crick base pairs, but where the nucleosides of one of the two strands will occur in an anticlinal conformation. Interestingly enough, the duplexes (L-RNA/D-RNA and L-DNA/D-RNA) in these models can show either right- or left-handedness. This is a very new observation, suggesting that molecular symmetry of enantiomeric nucleic acids is broken down.
| S-EPMC3906056 | biostudies-literature
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.
| S-EPMC6412130 | biostudies-literature
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.
| S-EPMC4464905 | biostudies-literature
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.
| S-EPMC108972 | biostudies-literature
Project description:AIM:To explore whether HDV ribozymes have the ability to trans-cleave HCV RNA. METHODS:Three HDV genomic ribozymes were designed and named RzC1, RzC2 and RzC3. The substrate RNA contained HCV RNA 5'-noncoding region and 5'-fragment of C region (5'-NCR-C). All the ribozymes and HCV RNA 5'-NCR-C were obtained by transcription in vitro from their DNA templates, and HCV RNA 5'-NCR-C was radiolabelled at its 5'-end. Under certain pH, temperature, appropriate concentration of Mg(2+) and deionized formamide, these ribozymes were respectively or simultaneously mixed with HCV RNA 5'-NCR-C and reacted for a certain time. The trans-cleavage reaction was stopped at different time points, and the products were separated with polyacrylamide gel electrophoresis (PAGE), displayed by autoradiography. Percentage of trans-cleaved products was measured to indicate the activity of HDV ribozymes. RESULTS:RzC1 and RzC2 could trans-cleave 26 % and 21.8 % of HCV RNA 5'-NCR-C under our reaction conditions with 2.5 mol.L(-1) deionized formamide respectively. The percentage of HCV RNA 5'-NCR-C trans-cleaved by RzC1, RzC2 or combined usage of the three ribozymes increased with time, up to 24.9 %, 20.3 % and 37.3 % respectively at 90 min point. Almost no product from RzC3 was observed. CONCLUSION:HDV ribozymes are able to trans-cleave specifically HCV RNA at certain sites under appropriate conditions, and combination of several ribozymes aiming at different target sites can trans-cleave the substrate more efficiently than using only one of them.
| S-EPMC4656322 | 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-literature
Project description:Although several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. Here, we report the isolation of two zinc-dependent DNAzymes, ZincDz1 and ZincDz2, which exhibit compact catalytic core sequences with highly versatile hydrolysis activity. They were selected through in vitro selection followed by deep sequencing analysis. Despite their sequence similarity, each DNAzyme showed different Zn2+-concentration and pH-dependent reaction profiles, and cleaved the target RNA sequences at different sites. Using various substrate RNA sequences, we found that the cleavage sequence specificity of ZincDz2 and its highly active mutant ZincDz2-v2 to be 5'-rN↓rNrPu-3'. Furthermore, we demonstrated that the designed ZincDz2 could cut microRNA miR-155 at three different sites. These DNAzymes could be useful in a broad range of applications in the fields of medicine and biotechnology.
| S-EPMC7886857 | biostudies-literature
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:BACKGROUND: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. RESULTS: We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and alpha-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two alpha-L-LNA monomers per binding arm (kobs increased from 0.014 min-1 to 0.78 min-1). CONCLUSION: The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers.
| S-EPMC1501032 | biostudies-literature