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Discovery and characterization of self-cleaving ribozyme structural variants

自裂解核酶结构变体的发现和表征

基本信息

批准号：
8729348
负责人：
Randi Jimenez
金额：
$ 3.62万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-08 至 2015-08-07
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8729348
关键词：
Advanced Development Automobile Driving Binding Binding Sites Biochemical Bioinformatics Biological Biological Assay Biological Process Biology Catalysis Catalytic Domain Catalytic RNA Cell Extracts Cleaved cell Computer software Conserved Sequence Data Databases Descriptor Development Diagnostic Disease Dissociation Drug Targeting Environment Family Functional RNA Future Gene Expression Genes Genome Genomics Health Hepatitis Delta Virus Human Biology Human Genome Human Microbiome In Vitro Incidence Knowledge Lead Life Ligand Binding Ligands Location Metagenomics Nature Outcome Pathway interactions Pattern Peripheral Phosphotransferases Play Process Publishing RNA Regulation Regulatory Pathway Research Research Project Grants Role Structure Testing Transcript Untranslated Regions Variant Work aptamer base cofactor computerized tools design divalent metal flexibility hammerhead ribozyme in vitro activity in vitro testing in vivo interest metagenomic sequencing novel public health relevance research study sensor small molecule tool

项目摘要

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.

描述（由申请人提供）：RNA发挥多种生物学作用，包括调节和催化作用。自切割RNA（核酶）的广泛分布强烈表明了其生物学意义。大多数自切割核酶的折叠和生物化学活性仅依赖于二价金属的存在，并且在大多数核酶家族中，没有已知的自切割调节机制。此外，核酶催化核心外围结构域的结构灵活性程度从未得到系统表征。本研究的目的是发现一种新的具有调节活性的自切割核酶。该目标将通过以下方式实现：1）使用能够搜索由用户定义的RNA结构基序的可用软件，2）创建新的计算工具以评估独特的外周结构域的保守性，3）测试推定的核酶在体外切割的活性，以及4）确定是否有任何辅因子正在调节核酶的活性。第一步是使用基于结构的搜索来鉴定两个自切割核酶家族的结构变体：锤头状核酶（HHR）和丁型肝炎病毒核酶（HDV）。这些计算搜索使用每个核酶家族的独特二级结构来指导通过可用的基因组序列数据库搜索能够折叠成相同基序的序列。第二步建立生物信息学工具，以评估预测的二级结构的保守性的核酶外围结构域。第三步通过体外催化活性测试来验证新核酶的发现。潜在核酶的调节将通过考虑活性核酶的保守结构变异和一致的基因组位置来推断。第四步将使用这些信息来预测可能参与调节核酶活性的候选小分子。将进一步分析最佳候选物的结合以及结合对自切割活性的影响。本研究的长期目标是是为了确定真核生物基因组中自切割核酶的生物学作用。这项研究将从两个核酶家族（HHR和HDV）开始，这两个家族广泛存在于所有生命领域，包括人类基因组中，但其生物学作用仍不清楚。拟议研究项目的实施将需要开发先进的计算工具，并继续开发用于RNA催化和配体结合的生化测定。揭示调节核酶活性的机制可能会导致发现新的调节途径。