Abstract
RNA is a highly versatile molecule that is involved in numerous aspects of gene expression including protein synthesis, RNA maturation, RNA editing, RNA turnover, translational regulation, and protein trafficking. A seminal discovery that significantly diversified the functional repertoire of RNA came in the early 1980s with the Nobel Prize-winning discovery that certain RNAs possess catalytic activity. RNA catalysts (ribozymes) abound in nature, possess a wide range of activities, and are involved in several aspects of gene expression. The multifunctional character of RNA has propelled the development of novel RNA- based therapeutic and diagnostic agents. Although ribozymes are highly versatile in such applications, their activities are not naturally subject to regulatory mechanisms. The ability to control ribozyme activity would be beneficial toward regulating the processes in which they are involved. Therefore, I am interested in engineering RNA catalysts that can be artificially regulated by the binding of specific ligands. Such allosteric ribozymes might serve as controllable molecular switches in both therapeutic and diagnostic applications. Toward the goal of developing ligand-dependent RNA catalysts and assessing their intracellular function, I have isolated and characterized a variety of allosteric ribozymes derived from the hammerhead and Hepatitis Delta Virus (HDV) ribozymes, as well as Tetrahymena group I self-splicing intron. The generation of allosteric ribozymes is achieved by functionally integrating ribozyme and ligand-binding RNA domains using rational design strategy and combinatorial selection techniques.|Through combinatorial selection of allosteric HDV ribozymes, a small RNA module was isolated that functions as a conditionally folded element capable of adjoining a variety of ligand-binding and ribozyme domains. The module not only provided an opportunity to examine the structure-function relationship between ligand binding and catalysis, but also enabled the generation of novel allosteric ribozymes. Using rational design, the module facilitated construction of allosteric Tetrahymena group I self-splicing introns. Certain allosteric self-splicing introns were demonstrated to function intracellularly, where one was further capable of regulating reporter gene expression in E. coli. Toward the goal of utilizing allosteric ribozymes to modulate mammalian gene expression, I explored whether ligand-dependent HDV ribozymes could regulate the production of small interfering RNAs (siRNAs) that function through the RNA interference (RNAi) pathway to down-regulate target gene expression. Although the allosteric ribozymes failed to regulate target gene expression, constitutively active or inactive ribozyme constructs demonstrated that ribozyme-mediated siRNA production and RNAi are fundamentally achievable. Taken together these studies demonstrate that the modular character of functional RNAs can be exploited to generate ligand-dependent ribozymes. Moreover, such catalysts are capable of functioning intracellularly in response to ligand, and can be utilized to regulate gene expression.