Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries

Abstract

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 ac ell-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 ; 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 selfcleavage.

Screening for engineered neomycin riboswitches that control translation initiation

“…In the last few years several engineered riboswitches have been developed that are based on either antisense strategies (Isaacs et al 2004;Bayer and Smolke 2005), regulatable ribozymes (Yen et al 2004(Yen et al , 2006, or small moleculebinding aptamers (Werstuck and Green 1998;Suess et al 2003). The latter control gene expression by insertion of aptamer sequences into the 59 untranslated region (UTR) of an mRNA.…”

Expanded hammerhead ribozymes containing addressable three-way junctions

“…The HHR has been shown to be a powerful and versatile tool for the in cis regulation of gene expression (Yen et al 2004(Yen et al , 2006Win and Smolke 2007a;Wieland and Hartig 2008). In order to broaden its applicability, additional ways of connecting the hammerhead to natural RNAs are highly warranted.…”

“…Reconstitution of gene expression was achieved by the addition of nucleoside analogs. Upon unspecific incorporation into the cellular RNA, the analogs interfere with ribozyme activity, resulting in full-length mRNA and restored gene expression levels (Yen et al 2004(Yen et al , 2006. However, the mechanism of this ribozyme switch is rather unspecific, potentially resulting in toxic side effects of the nucleoside analogs.…”

Artificial Riboswitches: Synthetic mRNA‐Based Regulators of Gene Expression

“…The underlying mechanism is based on the incorporation of the analogues into the mRNA, and consequently in the HHR sequence, whereby the cleavage activity is reduced (see Scheme 5 B). [34] However, the action of the nucleoside analogues is not ribozyme-specific and is likely to show cytotoxic side effects, especially in prokaryotes.…”

“…This leads to nonspecific inhibition of the HHR activity. [33,34] C) and D) Specific HHR activity control can be obtained by attaching aptamers to stem II. Ligand binding thereby affects essential tertiary interactions and finally ribozyme activity.…”