Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research最新文献_第7页

Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm. 选择性dnazyme通过靶向突变位点或通过结合臂的错配介导AChR突变转录物的切割。

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Pub Date : 2005-07-28

Amr Abdelgany, John Ealing, Matthew Wood, David Beeson

{"title":"Selective DNAzyme-mediated cleavage of AChR mutant transcripts by targeting the mutation site or through mismatches in the binding arm.","authors":"Amr Abdelgany, John Ealing, Matthew Wood, David Beeson","doi":"","DOIUrl":"","url":null,"abstract":"Many dominantly inherited disorders are caused by missense amino acid substitutions resulting from a single nucleotide exchange in the encoding gene. For these disorders, where proteins expressed from the mutant alleles are often pathogenic and present throughout life, gene silencing, through intervention at the mRNA level, holds promise as a therapeutic approach. We have used mutations that underlie the slow channel congenital myasthenic syndrome (SCCMS) as a model system to study allele-specific gene silencing of RNA transcripts by DNAzymes. We tested the ability of DNAzymes to give allele-specific cleavage for i) mutations that create cleavage sites, and ii) mutations located close to a DNAzyme cleavage site that create a potential mismatch in the binding arms. For both we demonstrate selective cleavage of mutant transcripts under simulated physiological conditions. For DNAzymes with binding arm mismatches the degree of selectivity for mutant over wild type may be enhanced by optimising the mismatch position as well as the binding arm length. The optimal sites for mismatches are 1.1 and 1.2 in arm I, and 16.2 in arm II. Asymmetric binding arm DNAzymes with a shorter arm I are more discriminative. Our results show it should be possible to apply DNAzyme-mediated cleavage of mutant alleles even when the mutant does not itself create a putative cleavage site. This therapeutic approach may be well suited to dominantly inherited disorders such as SCCMS, where loss of some wild type transcripts is unlikely to have pathogenic consequences.","PeriodicalId":88272,"journal":{"name":"Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research","volume":"1 1","pages":"32-7"},"PeriodicalIF":0.0,"publicationDate":"2005-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737196/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28416433","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Design and application of a versatile expression vector for RNAi in mammalian cells. 哺乳动物细胞中多功能RNAi表达载体的设计与应用。

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Pub Date : 2005-07-28

Xia Lin, Xin-Hua Feng

引用次数: 0

Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes. 锤头核酶选择性切割含有慢通道肌无力综合征突变的AChR crna。

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Pub Date : 2005-07-28

Amr Abdelgany, John Ealing, Matthew Wood, David Beeson

{"title":"Selective cleavage of AChR cRNAs harbouring mutations underlying the slow channel myasthenic syndrome by hammerhead ribozymes.","authors":"Amr Abdelgany, John Ealing, Matthew Wood, David Beeson","doi":"","DOIUrl":"","url":null,"abstract":"Slow channel congenital myasthenic syndrome (SCCMS) is a dominant disorder caused by missense mutations in muscle acetylcholine receptors (AChR). Expression from mutant alleles causes prolonged AChR ion-channel activations. This 'gain of function' results in excitotoxic damage due to excess entry of calcium ions that manifests as an endplate myopathy. The biology of SCCMS provides a model system to investigate the potential of catalytic nucleic acids for therapy in dominantly inherited disorders involving single missense mutations. Hammerhead ribozymes can catalytically cleave RNA transcripts in a sequence-specific manner. We designed hammerhead ribozymes to target transcripts from four SCCMS mutations, alphaT254I, alphaS226F, alphaS269I and epsilonL221F. Ribozymes were incubated with cRNA transcripts encoding wild type and mutant AChR subunits. The ribozymes efficiently cleaved the mutant allele cRNA transcripts but left the wild type cRNA intact. Cleavage efficiency was optimised for alphaS226F. We were able to demonstrate robust catalytic activity under simulated physiological conditions and at high Ca(2+) concentrations, which is likely to be accumulated at the endplate region of the SCCMS patient muscles. These results demonstrate the potential for gene therapy applications of ribozymes to specifically down-regulate expression of mutant alleles in dominantly inherited disorders.","PeriodicalId":88272,"journal":{"name":"Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research","volume":"1 1","pages":"26-31"},"PeriodicalIF":0.0,"publicationDate":"2005-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737193/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28416432","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

PCR-based expression analysis and identification of microRNAs. 基于pcr的microrna表达分析与鉴定。

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Pub Date : 2005-07-28

David P Lu, Rebecca L Read, David T Humphreys, Fiona M Battah, David I K Martin, John E J Rasko

{"title":"PCR-based expression analysis and identification of microRNAs.","authors":"David P Lu, Rebecca L Read, David T Humphreys, Fiona M Battah, David I K Martin, John E J Rasko","doi":"","DOIUrl":"","url":null,"abstract":"microRNAs (miRNAs) are small RNAs that regulate translation and hence control a variety of cellular processes in metazoans. The quantitation and identification of miRNAs has been hampered by their small size and low abundance. Here we describe two robust PCR-based assays of miRNA expression based on the original cloning strategy. The non-quantitative PCR method allows detection and identification of miRNAs and we utilise this method in the discovery of a new miRNA (miR-532) in retinoic acid differentiated P19 cells. The second and quantitative method (QM-RT-PCR) is simple and accurate, and uses commonly available technology. Of particular interest is the specificity of this PCR-based technology compared to hybridisation-based methods including arrays and northern blotting. Here we have shown that a single base pair mismatch in the priming sequence results in a two order of magnitude reduction in the amplification of let-7f. These streamlined methods will complement previously described methods and will facilitate analysis of miRNA expression in rare cell populations where the amount of RNA is limited.","PeriodicalId":88272,"journal":{"name":"Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research","volume":"1 1","pages":"44-9"},"PeriodicalIF":0.0,"publicationDate":"2005-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737195/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28416435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Vector-based siRNA delivery strategies for high-throughput screening of novel target genes. 基于载体的siRNA递送策略用于高通量筛选新的靶基因。

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Pub Date : 2005-07-27

Meihong Chen, Quan Du, Hong-Yan Zhang, Claes Wahlestedt, Zicai Liang

引用次数: 0

The RISC component VIG is a target for dsRNA-independent protein kinase activity in Drosophila S2 cells. RISC成分VIG是果蝇S2细胞中不依赖dsrna的蛋白激酶活性的靶标。

Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Pub Date : 2005-07-27

Konstantin I Ivanov, Timofey V Tselykh, Tapio I Heino, Kristiina Mäkinen

{"title":"The RISC component VIG is a target for dsRNA-independent protein kinase activity in Drosophila S2 cells.","authors":"Konstantin I Ivanov, Timofey V Tselykh, Tapio I Heino, Kristiina Mäkinen","doi":"","DOIUrl":"","url":null,"abstract":"RNA interference (RNAi) is mediated by a multicomponent RNA-induced silencing complex (RISC). Here we examine the phosphorylation state of three Drosophila RISC-associated proteins, VIG, R2D2 and a truncated form of Argonaute2 devoid of the nonconserved N-terminal glutamine-rich domain. We show that of the three studied proteins, only VIG is phosphorylated in cultured Drosophila cells. We also demonstrate that the phosphorylation state of VIG remains unchanged after cell transfection with exogenous dsRNA. A sequence similarity search revealed that VIG shares significant similarity with the human phosphoprotein Ki-1/57, a known in vivo substrate for protein kinase C (PKC). In vitro kinase assays followed by tryptic phosphopeptide mapping showed that PKC could efficiently phosphorylate VIG on multiple sites, suggesting PKC as a candidate kinase for VIG phosphorylation in vivo. Taken together, our results identify the RISC component VIG as a novel kinase substrate in cultured Drosophila cells and suggest a possible involvement of PKC in its phosphorylation.","PeriodicalId":88272,"journal":{"name":"Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research","volume":"1 1","pages":"12-20"},"PeriodicalIF":0.0,"publicationDate":"2005-07-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737197/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28416430","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0