Abstract The overall vision of the Bevilacqua lab is that human health can be advanced by combining holistic genomic and detailed molecular views of fundamental problems in RNA biology. Over the next five years, the work outlined in this MIRA will advance RNA folding and catalysis under in vivo and in vivo-like conditions using a combination of traditional and high-throughput (HT) experimental and computational approaches, many of which the lab has helped develop. There will be a balance of development of fundamental methods and new technologies with their application. The molecular perspective employs both traditional biophysical and biochemical approaches, while the holistic perspective uses next-generation sequencing (NGS) approaches. A major goal of this MIRA proposal is to connect these two approaches to discover novel RNA structures and functions and to understand them from both gene regulatory and molecular perspectives. This proposal brings multiple high-throughput (HT) approaches to bear including experimental approaches such as fluorescence-detected binding isotherms (FDBI), massively parallel oligonucleotide synthesis (MPOS), and Structure-seq NGS, as well as computational approaches ranging from cheminformatics to bioinformatics. Goals for the next five years encompass probing RNA folding under biological conditions that include deriving an expanded set of nearest neighbor (NN) parameters for describing RNA folding in the presence of bacterial and human metabolite mixtures. The lab will also explore the dynamics of RNA folding in such in vivo-like conditions using time-resolved studies such as fluorescence-detected stopped-flow kinetics, as well as temperature-dependent probing of RNA structure in vivo to uncover cases where only a fraction of the RNA molecules form a given pairing. Additional efforts will focus on discovery of and novel functions for ribozymes including tolerance of mutation, metabolite assistance of cleavage, occurrence of zymogens (‘r