The discovery that nucleic acids mediated the inhibition of gene expression in a sequence-specific manner has provided the scientific community with a potentially important tool to analyse gene function and validate drug targets. Selective inhibition of gene expression by ribozymes and small interfering RNAs siRNAs is being explored for potential therapeutics against viral infections, inflammatory disorders, haematological diseases and cancer. In order to be used as pharmaceutical drugs, chemical modifications are necessary to increase their stability in vivo. To attain stability, ribozymes and siRNAs must also overcome several other problems, including accessibility to target messenger RNAs mRNAs , efficient delivery to target cells and unwanted non-specific effects.
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Potential Design Rules and Enzymatic Synthesis of siRNAs
This is a preview of subscription content, log in to check access. Blood — PubMed Google Scholar. Oncologist — PubMed Google Scholar. Their optimized techniques employ hairpin ribozymes, DNAzymes, hammerhead ribozymes and derivatives, group I intron ribozymes, RNase P ribozymes, and siRNAs, as well as general methods for RNA structure analysis, delivery of oligonucleotides, and gene therapy. Also provided are novel methods for identifying accessible cellular mRNA sites; group I intron and RNAse P ribozyme protocols for effective design, selection, and therapeutic applications; and the latest RNAi methods for sequence-specific gene silencing in a wide variety of organisms.
Additional techniques cover the analysis of ribozyme structures and conformational transitions using nucleotide analog interference mapping and fluorescence resonance energy transfer, the use of ribozymes in clinical and gene therapy, and the use of ribozymes and DNAzymes in rodent models of human disease. Each proven protocol includes a background introduction outlining the principle behind the technique, step-by-step instructions, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and up-to-date, Ribozymes and siRNA Protocols details for experienced and novice investigators alike the many exciting advances in our understanding of nucleic acid enzymes, as well as demonstrating how they may be used to analyze gene function and target validation, and to productively develop novel therapeutics for human diseases.
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Book Description Condition: New. US edition. Perfect condition. Customer satisfaction our priority. No off-target effect was detected for any of the corresponding saiRNAs Fig. We constructed reporter plasmids containing an overlapping region of mRNAs and their antisense transcripts inserted downstream of a firefly luciferase gene in the forward sense target or reverse antisense target orientation. Other genes may have been downregulated due to the off-target effects by non-canonical base pairing between siRNAs and target mRNAs or due to secondary effects.
To confirm the results of the transcriptome analysis, we randomly selected 14 genes significantly downregulated by off-target effects Supplementary Fig. Good correlation with the transcriptome data was observed Supplementary Fig. Cells transfected with a control vector containing only the HDV ribozyme displayed no such changes, indicating that the ribozyme itself had no influence Supplementary Fig. Taken together, these results indicate that saiRNA induces fewer off-target effects on endogenous genes than shRNAs or their modified versions.
The nucleotides in blue indicate the sequence of the H1 promoter.
Previous studies have shown that the sustained overexpression of shRNA interfered with the processing and exporting of endogenous miRNAs Interestingly, the relative abundance of individual miRNAs compared with total miRNAs did not change in the correlation analysis, suggesting that the decrease in the proportion of total miRNAs on shRNA overexpression is most likely mediated by competition for the general processing and export machinery of miRNAs Fig. These results indicate that saiRNA has less influence on the biogenesis of endogenous miRNAs, which is consistent with their unique Dicer-independent biogenesis pathway and the lower accumulation of siRNAs associated with Ago proteins.
In this study, we have investigated shRNAs with different structural features and designed a new potent siRNA precursor enhanced by a ribozyme. The siRNA precursors can be divided into three categories according to their processing pathway, which is mainly determined by stem length. This design significantly increases the accumulation of siRNAs and improves the silencing efficiency of saiRNA to a level comparable to or better than that of the classical shRNAs.
However, we cannot rule out the possibility that such selectivity is assisted by other cofactors that interact with Ago2. The specific processing pathway of saiRNA has several advantages that enhance both the efficiency and specificity of RNAi.
By contrast, the strand-specific cleavage of saiRNA by Ago2 during its processing completely eliminates the passenger strand and reduces off-target effects. Importantly, transcriptome analyses have revealed the existence of numerous antisense transcripts that may have important regulatory functions 47 , 48 , The single-strand feature of saiRNA could be especially useful for studying gene function without the risk of unintentional cleavage of antisense transcripts and lncRNAs mediated by the siRNA passenger strand, which may have unpredictable consequences. This result is likely due to the exclusive association of siRNAs generated from saiRNAs with Ago2 but not the other three non-nucleolytic Ago proteins 32 , Although the rules for the design of effective saiRNAs are still elusive, several high-throughput in vitro screening methods for highly potent RNAi triggers might be modified to identify the most effective saiRNAs and describe general principles for the design of saiRNAs 59 , For shRNA, both the guide strand carmine and the passenger strand blue of the siRNA are produced by Dicer cleavage and are incorporated into all Ago proteins.
Both the strands associated with Ago1, 3 and 4 can cause off-target effects. The passenger strand associated with Ago2 not only mediates off-target effects but also cleaves the antisense transcript of target mRNA with possibly unintended consequences. Ago1, 3 and 4 are incapable of processing saiRNA due to their lack of endonucleolytic activity and do not cause any off-target effects.
The reporter plasmids used in Supplementary Fig.
All the primers are listed in Supplementary Methods. Mycoplasma contaminations were regularly tested for all the cell lines. The cells were harvested at different time points for RNA extraction and Northern blotting analysis. All the uncropped scans of the Northern blots are shown in Supplementary Fig. All the uncropped scans of the western blots are shown in Supplementary Fig.
The data were analysed with StepOne software v2. The virus titres were calculated according to a standard curve generated by using a series of diluted standard virus samples Applied Biological Materials Inc. The raw fastq data were pre-processed using a common procedure. For the correlation assay of miRNA expression in Fig. The human transcriptome annotation was retrieved from Gencode version The human genome was downloaded from UCSC version hg How to cite this article: Shang, R.
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Knockdown of Telomerase RNA Using Hammerhead Ribozymes and RNA Interference
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