The post-genomic era has ushered in a profound new appreciation of the broad and central roles played by RNAs across the cell. We now face the challenge of elucidating the underlying functional mechanisms of RNAs to truly understand, engineer, and correct their function in biological systems and disease. However, our understanding of the fundamental sequence-structure-function relationship underlying RNA's role in life's most basic processes is still in its infancy because of the technical challenge of interrogating RNA structures in the dynamic, non-equilibrium environment of the cell. This is further confounded by a lack of high throughput tools that can characterize RNA structures on an 'omics'scale. Therefore, the scientific objective of this proposal is to address both of these challenges by developing an 'omics'technology that can determine the dynamic functional states of RNAs across the genome. We recently made a breakthrough step in this direction with our development of a high-throughput RNA structure characterization technology. This technology combines RNA structure chemical probing and next-generation sequencing to probe the structures of hundreds of RNAs simultaneously in vitro. Here, we seek to extend this technology to characterize the dynamic, co-transcriptional folding pathways of RNAs, and elucidate the extent to which an RNA's function is determined by the folding pathway it undergoes as it is actively transcribed. Our innovative approach to uncovering the dynamic folding pathways of RNA molecules turns the problem on its head: rather than monitor the folding processes of individual RNA molecules over time, we instead take snapshots of entire populations of RNA molecules and statistically reconstruct their folding trajectories. Our innovative technology is thus a creative combination of the throughput and sensitivity of next- generation sequencing, the versatility of chemical RNA structure probing, and the power of statistics to create a new approach to elucidate RNA structures and interactions formed during their folding pathways for the first time. This technology will be developed in the context of asking fundamental questions about the differences between equilibrium and co-transcriptional RNA folds, how ligands interact with RNAs during transcription and guide their folding pathways, and how nascent RNA folding couples to and even regulates transcription dynamics. We anticipate the outcome of this work will be a transformation in the way we think about the RNA structure-function relationship, thereby creating a new paradigm in our understanding of how RNA molecules perform ubiquitous, versatile and critical roles in life's most fundamental processes.