Yen Laising, Magnier Maxime, Weissleder Ralph, Stockwell Brent R, Mulligan Richard C
Department of Genetics, Harvard Medical School, and Division of Molecular Medicine, Children's Hospital, Boston, Massachusetts 02115, USA.
RNA. 2006 May;12(5):797-806. doi: 10.1261/rna.2300406. Epub 2006 Mar 23.
We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.
我们最近描述了一种用于哺乳动物细胞的仅基于RNA的基因调控系统，其中携带核酶序列的mRNA自我切割的抑制为基因表达的控制提供了基础。通过证明一种特定的RNA自我切割小分子抑制剂丰加霉素在体外和体内控制基因表达的能力，为该系统提供了一个重要的原理证明。在这里，我们描述了高通量筛选（HTS）分析方法的开发过程，该方法导致了丰加霉素和其他能够抑制哺乳动物细胞中RNA自我切割的分子的鉴定。为了鉴定可作为核酶自我切割抑制剂的小分子，我们建立了一种基于细胞的分析方法，其中荧光素酶（luc）报告基因的表达由核酶序列控制，并筛选了58076种化合物诱导荧光素酶表达的能力。通过该筛选鉴定出15种能够抑制细胞内核酶自我切割的化合物。鉴定出的最有效的抑制剂是丰加霉素和5-氟尿苷（FUR），它们是嘌呤或嘧啶碱基7位和5位带有修饰的核苷类似物。单独使用时，这两种化合物分别能够诱导核酶控制的报告基因的基因表达约365倍和110倍。对核酶抑制剂作用机制的研究表明，这些化合物必须掺入RNA中才能抑制RNA自我切割。
