MicroRNAs (miRNAs) are genomically-encoded small RNAs (~ 21 nts) that bind to target mRNAs in a sequence-specific manner to regulate their translation and/or decay. In addition to its role in regulating cholesterol and fatty acid metabolism, the abundant liver-specific microRNA miR-122 also plays a central role in the pathogenesis of Hepatitis C virus (HCV) and associated hepatocellular carcinoma by stimulating viral replication through its binding to target sites in the 5'noncoding region of HCV RNA. A number of antisense approaches, which involve the repeated high-dose administration of modified oligonucleotides, have been described for antagonizing miR-122 function. This proposal describes a novel approach using a sequence-specific ribozyme to inactivate miR-122. The advantage of a ribozyme is that it is catalytic;once introduced into the cell, a single molecule will inactivate multiple copies of the miRNA target. Our research design exploits the properties of M1 RNA, the catalytic subunit of Escherichia coli RNase P. The covalent attachment of a guide sequence to M1 RNA generates a customized ribozyme that selectively inactivates the complementary target RNA. We have recently demonstrated the usefulness of this approach to disrupt miRNA function in Arabidopsis plants. Aim 1 will employ both RNA engineering and in vitro evolution strategies to develop an array of M1 RNA-based ribozymes that will effectively and selectively degrade miR-122. Aim 2 will evaluate the efficacy of these ribozymes against miR-122 in Huh-7 liver cancer cells, which have high levels of miR-122. To control the level of ribozyme, it will be expressed from a tetracycline-regulated promoter. Establishing the quantitative relationship between ribozyme expression, the levels of miR-122 and target gene expression will be a cornerstone of this work. RNase protection and qPCR assays will be used to measure the levels of ribozymes and miR-122, respectively. Target gene expression will be monitored using a Renilla luciferase reporter with three miR-122 binding sites from the cationic amino acid transporter 1 (CAT-1) mRNA, qPCR for five endogenous target mRNAs, and by Western blotting for each of these proteins. miRNA microarrays will be used to determine specificity of the ribozyme for targeting miR-122. Lastly, the generality of this approach will be tested by replacing the miR-122 guide sequence in the ribozyme with one complementary to let-7a and thereby targeting let-7a. The overall goal of this R21 proposal is to determine the feasibility of disrupting miR-122 function with a ribozyme, and to lay the groundwork for a subsequent R01 application that will use the tools developed here to directly target HCV. PUBLIC HEALTH RELEVANCE: The replication of Hepatitis C virus (HCV) in liver cells is stimulated by the binding of microRNA-122 (miR-122), the most abundant small noncoding RNA in liver. This proposal describes a new approach to inactivating miR-122 using a catalytic RNA enzyme. The long term goal is to apply this strategy to the treatment of HCV infection.