Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa, Japan.
Methods Mol Biol. 2021;2323:213-220. doi: 10.1007/978-1-0716-1499-0_15.
This chapter describes a screening strategy to engineer synthetic riboswitches that can chemically regulate gene expression in mammalian cells. Riboswitch libraries are constructed by randomizing the key nucleotides that couple the molecular recognition function of an aptamer with the self-cleavage activity of a ribozyme. The allosteric ribozyme (aptazyme) candidates are cloned in the 3' untranslated region (UTR) of a reporter gene mRNA. The plasmid-encoded riboswitch candidates are transfected into a mammalian cell line to screen for the desired riboswitch function. Furthermore, multiple aptazymes can be cloned into the 3' UTR of a desired gene to obtain a logic gate response to multiple chemical signals. This screening strategy complements other methods to engineer robust mammalian riboswitches to control gene expression.
本章描述了一种筛选策略，用于设计能够在哺乳动物细胞中化学调控基因表达的合成核糖开关。核糖开关文库通过随机化连接适体的分子识别功能与核酶自身切割活性的关键核苷酸来构建。变构核酶（aptazyme）候选物被克隆到报告基因 mRNA 的 3'非翻译区 (UTR)。将质粒编码的核糖开关候选物转染到哺乳动物细胞系中，以筛选所需的核糖开关功能。此外，多个 aptazyme 可以被克隆到所需基因的 3'UTR 中，以获得对多个化学信号的逻辑门响应。这种筛选策略补充了其他设计稳健的哺乳动物核糖开关以控制基因表达的方法。
