In contrast with the earliest models suggesting that it functioned only as messenger, RNA is now known to participate in a myriad of essential biological processes, ranging from gene expression through protein manufacturing. Short double-stranded RNAs participate in selective degradation of messenger RNA transcripts (RNA interference); folded RNAs can catalyze reactions, or switch genes on and off; in conjunction with numerous proteins, RNAs fold into large molecular machines, like the protein-manufacturing ribosome. Furthermore, because small RNAs can be designed to selectivity bind specific targets, these molecules have enormous potential as therapeutics. These widely varying roles are enabled by RNA's structural versatility. We propose to test new hypotheses about the roles of ions and RNA architecture in RNA self-assembly or folding. We will use time resolved small angle x-ray scattering to report the time-dependent formation of global structures, time-resolved hydroxyl radical footprinting to report the formation of individual contacts within the folding molecule, and time-resolved fluorescence methods to report specific intramolecular distances during folding. To enhance experimental studies, we propose to develop efficient atomically detailed simulations of the kinetics and thermodynamics of RNA folding. Initially, these coupled tools will be applied to further quantify the important role of ions in RNA folding. We will also study folding of different classes of RNA enzymes or ribozymes to explore the role of RNA architecture in folding. Our long term goal is to understand the factors involved in RNA folding. Such knowledge is essential for the efficient development and application of RNA-based therapeutics. PUBLIC HEALTH RELEVANCE: RNA molecules can fold into biologically active structures that carry out a wide variety of functions. The numerous roles assumed by RNA highlight its usefulness and its great potential as a therapeutic molecule or a target for other therapeutics. These studies will enable us to better understand how RNA structures assemble, thus will teach us how to more efficiently design RNAs for application in medicine or biotechnology.