Development of in Vitro Genetic System to Study RNA Editing

本研究的目的是利用体外选择作为模型系统来探索RNA催化和着丝质体RNA编辑之间的可能联系。RNA编辑是在一些最早的真核生物，即着丝质体原生动物的线粒体中发生的奇怪而复杂的尿嘧啶残基的添加或删除。该方法将是开发一个成功的体外选择系统来分析RNA编辑的机制和进化。这些实验将解决古老的RNA编辑和RNA催化剂在进化中的作用。一种假设是，RNA编辑是一个古老的过程，几个短基因座相互作用产生一个最终转录本可能反映了基因首次组装的一些步骤。具体来说，这些实验将通过分析RNA编辑的每个步骤是否可以被RNA催化（这是一些最早假设的生物机制的特征），来验证RNA编辑可能是RNA世界中的关键角色之一的假设。这项研究将确定RNA编辑的第一步，即类似于其他RNA催化的连接反应的酯交换反应，是否可以由RNA分子催化。通过一系列体外选择实验，模拟试管中的进化，实验将通过从随机序列池中选择所需的催化分子，探索可能的原始RNA编辑酶的世界。有了这些初步实验，我们将准备分析RNA编辑的后续步骤，最终目标是在体外进化出完整的RNA催化RNA编辑系统。这些实验将测试一个能够编辑RNA的分子是否可以在相当简单的条件下产生：一个复杂分子过程进化的模型。最终，这将允许人们设计出新的RNA酶，可以编辑任何特定序列的RNA。本研究的目的是利用体外筛选来探索RNA催化与着丝质体RNA编辑之间的可能联系。RNA编辑是在一些最早的真核生物，即着丝质体原生动物的线粒体中发生的奇怪而复杂的尿嘧啶残基的添加或删除。该方法将是开发一个成功的体外选择系统来分析RNA编辑的机制和进化。这些实验将测试一个能够编辑RNA的分子是否可以在相当简单的条件下产生：一个复杂分子过程进化的模型。通过一系列在试管中模拟进化的体外选择实验，这些实验将通过从随机序列池中选择新的催化分子来探索可能的原始RNA编辑酶的世界。最终，这将允许人们设计出新的RNA酶，可以编辑任何特定序列的RNA。＊＊＊

9520253 Landweber The objective of this research is to develop the use of in vitro selection as a model system to explore he possible link between RNA catalysis and kinetoplastid RNA editing. RNA editing is the bizarre and complex addition or deletion of uridine residues which occurs in the mitochondria of some of the earliest eukaryotes, the kinetoplastid protozoa. The approach will be to develop a successful in vitro selection system to analyze the mechanism and evolution of RNA editing. These experiments will address the antiquity of RNA editing and the role of RNA catalysts in evolution. One hypothesis is that RNA editing is an ancient process, and the interaction of several short loci to produce one final transcript may reflect some of the steps involved in the first assembly of genes. Specifically, these experiments will test the hypothesis that RNA editing may have been one of the key players in the RNA world by analyzing whether each of its steps can be catalyzed by RNA, a characteristic of some of the earliest hypothesized biological mechanisms. This research will determine whether the first step of RNA editing, a transesterification similar to other RNA catalyzed ligation reactions, can be catalyzed by an RNA molecule. Through a series of in vitro selection experiments, which simulate evolution in a test tube, the experiments will explore the world of possible primitive RNA editing enzymes, by selecting the desired catalytic molecules from a pool of random sequences. Armed with these preliminary experiments, we will then be prepared to analyze the later steps of RNA editing, with the final goal of evolving a complete RNA catalyzed RNA editing system in vitro. These experiments will test whether a molecule capable of editing RNA could have arisen under fairly simple conditions: a model for the evolution of complex molecular processes. Ultimately, this will allow one to design new RNA enzymes that can edit RNA of any specific sequence. %%% The objective od this research is to deve lop the use of in vitro selection to explore the possible link between RNA catalysis and kinetoplastid RNA editing. RNA editing is the bizarre and complex addition or deletion of uridine residues which occurs in the mitochondria of some of the earliest eukaryotes, the kinetoplastid protozoa. The approach will be to develop a successful in vitro selection system to analyze the mechanism and evolution of RNA editing. These experiments will test whether a molecule capable of editing RNA could have arisen under fairly simple conditions: a model for the evolution of complex molecular processes. Through a series of in vitro selection experiments, which simulate evolution in a test tube, the experiments will explore the world of possible primitive RNA editing enzymes, by seleating new catalytic molecules from a pool of random sequences. Ultimately, this will allow one to design new RNA enzymes that can edit RNA of any specific sequence. ***