In Vitro Evolution of RNA Modifying Ribozymes from Random Sequence

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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.