{"title":"Production of Recombinant RNA in Escherichia coli Using Eggplant Latent Viroid as a Scaffold.","authors":"Beltrán Ortolá, José-Antonio Daròs","doi":"10.1007/978-1-0716-1464-8_25","DOIUrl":null,"url":null,"abstract":"<p><p>Similar to viruses, viroids can also be engineered and transformed into useful biotechnological tools. We describe here a viroid-based system to produce large amounts of recombinant RNA in Escherichia coli. A precursor of eggplant latent viroid (ELVd), with the RNA of interest inserted between positions U245 and U246, is co-expressed in E. coli along the chloroplastic isoform of the eggplant tRNA ligase, the enzyme that mediates the circularization of this viroid in the infected plants. In the bacterial cells, the chimeric ELVd-RNA-of-interest precursor self-cleaves through the embedded hammerhead ribozymes, and the monomer is recognized and circularized by the co-expressed tRNA ligase. The resulting circular RNA, likely bound to the tRNA ligase, accumulates to a high concentration in the bacterial cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2316 ","pages":"315-327"},"PeriodicalIF":0.0000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in molecular biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1007/978-1-0716-1464-8_25","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 1
Abstract
Similar to viruses, viroids can also be engineered and transformed into useful biotechnological tools. We describe here a viroid-based system to produce large amounts of recombinant RNA in Escherichia coli. A precursor of eggplant latent viroid (ELVd), with the RNA of interest inserted between positions U245 and U246, is co-expressed in E. coli along the chloroplastic isoform of the eggplant tRNA ligase, the enzyme that mediates the circularization of this viroid in the infected plants. In the bacterial cells, the chimeric ELVd-RNA-of-interest precursor self-cleaves through the embedded hammerhead ribozymes, and the monomer is recognized and circularized by the co-expressed tRNA ligase. The resulting circular RNA, likely bound to the tRNA ligase, accumulates to a high concentration in the bacterial cells.
期刊介绍：
For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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