Instituto de Biología Molecular y Celular de Plantas (Consejo Superior de Investigaciones Científicas-Universitat Politècnica de València), Valencia, Spain.
Methods Mol Biol. 2022;2316:315-327. doi: 10.1007/978-1-0716-1464-8_25.
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.
类似于病毒，类病毒也可以被设计和转化为有用的生物技术工具。我们在这里描述了一种基于类病毒的系统，可以在大肠杆菌中大量生产重组 RNA。茄子潜隐病毒 (ELVd) 的前体与感兴趣的 RNA 插入到位置 U245 和 U246 之间，与茄子 tRNA 连接酶的叶绿体同工型一起在大肠杆菌中表达，该酶介导该类病毒在受感染植物中的环化。在细菌细胞中，嵌合的 ELVd-RNA 前体通过嵌入的锤头核酶自我切割，单体被共表达的 tRNA 连接酶识别并环化。所得的环状 RNA，可能与 tRNA 连接酶结合，在细菌细胞中积累到很高的浓度。
