Discovery and characterization of self-cleaving ribozyme structural variants

描述（申请人提供）：RNA具有多种生物学作用，包括调控和催化作用。自切割rna（核酶）的广泛分布强烈表明其生物学意义。大多数自裂核酶的折叠和生化活性仅依赖于二价金属的存在，并且在大多数核酶家族中没有已知的自裂调节机制。此外，核酶催化核心周围结构域的结构灵活性程度尚未系统表征。本研究的目的是揭示一种具有调节活性的自裂核酶的新发生率。目标将通过以下方式实现：1)使用能够搜索用户定义的RNA结构基序的可用软件，2)创建新的计算工具来评估独特外周结构域的保护，3)测试用于体外切割的假定核酶的活性，以及4)确定是否有辅助因子

DESCRIPTION (provided by applicant): RNA plays diverse biological roles, including in regulation and catalysis. The widespread distribution of self-cleaving RNAs (ribozymes) strongly suggests a biological significance. The folding and biochemical activity of most self-cleaving ribozymes are only dependent upon the presence of divalent metals and in a majority of ribozyme families there is no known regulatory mechanism of self-scission. In addition, the extent of structural flexibility in the domains peripheral to the ribozyme catalytic cores has neve been systematically characterized. The objective of this study is to uncover a new incidence of a self-cleaving ribozyme with regulated activity. The objective will be accomplished by: 1) using available software capable of searching for RNA structural motifs defined by the user, 2) creating new computational tools to assess the conservation of unique peripheral domains, 3) testing the activity of putative ribozymes for cleavage in vitro, and 4) determine if any cofactors are regulating ribozyme activity. The first step is to use structure-based searches to identify structural variants for two self-cleaving ribozyme families: hammerhead ribozyme (HHR) and hepatitis delta virus ribozyme (HDV). These computational searches use the unique secondary structure of each ribozyme family to guide a search through available genomic sequence databases for sequences capable of folding into the same motif. The second step builds bioinformatics tools to assess the conservation of predicted secondary structures in the ribozyme peripheral domains. The third step verifies the discovery of new ribozymes by testing for catalytic activity in vitro. Regulation of potential ribozymes will be inferred by taking into consideration conserved structural variations and consistent genomic locations of active ribozymes. The fourth step will use this information to predict candidate small molecules which may be involved in regulating ribozyme activity. The best candidates will be analyzed further for binding and the effect of binding on self-cleavage activity. The long-term objective of this study is to identify the biological roles of self-cleaving ribozymes in eukaryotic genomes. The study wil begin with two families of ribozyme (HHR and HDV) which are widespread throughout all kingdoms of life, including in the human genome, but for which the biological roles remain unclear. The implementation of the proposed research project will require the development of advanced computational tools and continued development of biochemical assays for RNA catalysis and ligand binding. Uncovering a mechanism regulating ribozyme activity may lead to the discovery of new regulatory pathways.