In Vitro Evolution of RNA Modifying Ribozymes from Random Sequence

体外选择（IVS）或定向分子进化，允许分离和扩增通过选择标准的罕见序列。它既可以用来修饰现有的核酶，也可以从一个庞大的异质库中分离出新的核酶。本研究的目的是利用选择系统探索RNA催化、RNA编辑和修饰之间的可能联系。实验旨在测试RNA催化在益生元进化中的作用，以及各种形式的RNA加工和修饰的古老，并探索具有修饰RNA能力的核酶的进化。通过从大约1016个随机序列的不同池中选择和表征新型核酶，将研究益生元系统的RNA催化范围和“催化任务空间”。一个特定的目标是从随机的RNA序列中分离出新的2'-o-甲基化核酶，这些RNA序列含有一个结合甲基供体作为辅助因子的核心。分离甲基化核酶的方法是基于含有单个2'-o-甲基核糖的寡核苷酸对碱水解的抗性，作为分离甲基化与非甲基化核糖核苷酸底物及其相关核酶的手段。另一个目的是分析由IVS发现的一种核酶（在先前的NSF SGER支持下），该核酶催化一种新的模板定向连接，涉及连接部位的非规范GU和G-G相互作用。这种意想不到的RNA催化连接的出现表明，它可能参与了从低核糖核苷酸前体合成更长的RNA模板的益生元合成。***最近从大量随机寡核苷酸序列中分离出新的核糖酶（RNA催化剂）的能力的证明，激起了人们对揭示RNA进化早期途径的可能性的兴奋，甚至复活可能已经灭绝超过30亿年的核糖酶活性。每1015个拷贝中就有一个分子存在，这些分子具有新的催化特性，甚至可以作为潜在的治疗剂，现在可以通过体外遗传学的新方法来检索。因此，通过组合化学或探索复杂的益生元途径的逐步进化，如自我复制或编辑RNA的能力，可以获得一种奇妙的工具来合成新型催化剂。本研究利用这一概念和方法的出现，通过实验设计来探索RNA进化，特别是具有修饰RNA能力的核酶。RNA催化在益生元进化中的作用以及各种形式的RNA加工和修饰的古代也将被研究。

9604377 Landweber In vitro selection (IVS) or directed molecular evolution, allows the isolation and amplification of rare sequences that pass a selection criterion. It can be used both to modify existing ribozymes and to isolate novel ones from a large heterogeneous pool. the objective of this research is to use the selection system to explore the possible link between RNA catalysis, RNA editing and modification. Experiments are designed to test the role of RNA catalysis in prebiotic evolution and the antiquity of a variety of forms of RNA processing and modification, and to explore the evolution of ribozymes that have the ability to modify RNA. The scope of RNA catalysis and "catalytic task space" available to prebiotic systems will be investigated by selecting and characterizing novel classes of ribozymes from different pools of approximately 1016 random sequences. One specific aim is to isolate novel 2'-o-methylating ribozymes from random RNA sequences containing a core which binds the methyl donor as a cofactor. The approach is isolating a methylating ribozyme is based on the resistance of an oligonucleotide containing a single 2'-o-methyl ribose to alkali hydrolysis as a means of separating methylated from unmethylated ribonucleotide substrates and their associated ribozymes. As additional aim is to analyze a ribozyme, discovered by IVS (under prior NSF SGER support), that catalyzes a novel template-directed ligation involving non-canonical GU and G-G interactions at the ligation site. The emergence of this unexpected RNA-catalyzed ligation suggests that is could have been involved in the prebiotic synthesis of longer RNA templates from oligoribonucleotide precursors. *** The recent demonstration of the ability to isolate new ribozymes (RNA catalysts) from a large pool of random oligonucleotide sequences has fueled an excitement about the possibility of uncovering early pathways of RNA evolution, or even resuscitating ribozyme activities that may have been exti nct for over three billion years. Molecules present in as few as one in 1015 copies, which have noval catalytic properties and could even serve as potential therapeutic agents, can now be retrieved by the new approach of in vitro genetics. Thus a fantastic tool is available for the synthesis of novel catalysts through combinatorial chemistry or probing the stepwise prebiotic evolution of complex prebiotic pathways, such as the ability to self-replicate or to edit RNA. This study takes advantage of this advent in concept and methodology, with experiments designed to explore RNA evolution, particularly the ribozymes that have the ability to modify RNA. The role of RNA catalysis in prebiotic evolution and the antiquity of a variety of forms of RNA processing and modification will also be studied.