An Unbiased Chemical Approach for the Discovery of Naturally Occurring Ribozymes

描述（由申请人提供）：RNA可以催化化学反应的发现揭示了这些分子的一种新作用，这种作用超出了遗传信息的传递。迄今为止，只有少数生物催化RNA（核酶）已被确定。这可能是由于缺乏无偏见的、基于活动的方法来识别它们。不依赖于二级结构预测的方法， 选择具有增强的反应性的RNA可以揭示新的核酶。 该提案描述了一种化学方法的发展，以发现生物核酶。这种基于活性的方法采用设计用于共价标记具有增强的化学反应性的RNA的小分子探针。设计核酶特异性化学探针的策略依赖于充分验证的假设，即核酶采用与蛋白质催化剂类似的工具;大多数核酶的三级结构导致具有增强的亲核性的官能团，其在很大程度上负责催化。设计用于选择性标记高度亲核RNA的适当设计的亲电化合物（基于活性的探针）将配备有亲和标签（生物素），并应用于从源自一系列细菌和真核生物的基因组的RNA库中选择异常反应性RNA。任何新发现的RNA的化学反应性和生物学作用将得到充分表征。 公共卫生相关性：近年来，发现了具有广泛新功能的RNA;这些分子现在被理解为执行许多曾经被认为仅限于蛋白质的相同功能，并且已经发现某些类别的RNA广泛存在于人类细胞中，在广泛的生物过程中发挥重要作用。一类特殊的RNA可能在细胞中具有广泛的重要性，但没有直接的检测方法存在，这是可以催化化学反应的RNA，通常称为核酶。该提案描述了一种新的化学方法的发展，以识别以前未知的天然核酶类别，我们希望这将导致对细胞功能的理解增加，并进而揭示治疗疾病的新靶点。

DESCRIPTION (provided by applicant): The discovery that RNA can catalyze chemical reactions revealed a novel role for these molecules that extends beyond the transfer of genetic information. To date, only a small number of biologically occurring catalytic RNAs (ribozymes) have been identified. This may be a result of the absence of unbiased, activity- based methods for their identification. Approaches that do not rely on secondary structure prediction and instead select RNAs possessing enhanced reactivity could reveal novel ribozymes. This proposal describes the development of a chemical approach to the discovery of biological ribozymes. This activity-based approach employs small-molecule probes designed to covalently label RNAs possessing enhanced chemical reactivity. The strategy for the design of ribozyme-specific chemical probes relies on the well-validated assumption that ribozymes employ similar tools as protein catalysts; the tertiary structures of most ribozymes result in functional groups with enhanced nucleophilicity that are largely responsible for catalysis. Appropriately designed electrophilic compounds (activity-based probes) designed to selectively label highly nucleophilic RNAs will be equipped with an affinity tag (biotin) and applied to the selection of unusually reactive RNAs from RNA pools derived from the genomes of a range of bacterial and eukaryotic organisms. The chemical reactivity and biological roles of any newly identified RNAs will be fully characterized. PUBLIC HEALTH RELEVANCE: In recent years, RNAs with a wide range of novel functions have been discovered; these molecules are now understood to perform many of the same functions that were once thought to be limited to proteins, and some classes of RNAs have been found to be widespread in human cells, playing vital roles in a broad range of biological processes. One particular class of RNAs that may have widespread importance in cells but for which no straightforward method of detection exists are RNAs that can catalyze chemical reactions, commonly referred to as ribozymes. This proposal describes the development of a new chemical method to identify previously unknown classes of naturally occurring ribozymes, which we hope will lead to an increased understanding of how cells function, and in turn reveal new targets for the treatment of disease.