RNA plays a central role in molecular biology, mediating and regulating the genetic code of DNA to yield proteins. This role linking code and functionality gives RNA directed design tremendous potential. Surprisingly, the dynamic nature and regulatory roles of RNA are driven by interactions with fundamental partners: metal ions and small molecules. A biophysical understanding of these interactions is key to predictive RNA re-engineering. Here we combine computational and microfluidic tools to elucidate principles that link RNA function to interactions with these fundamental elements. In the first half of the dissertation, we probe the role of divalent metal ions in mediating RNA folds. Ion counting and structural modelling methods elucidate unique ionic signatures in RNA that fundamentally explains the disparity in roles between RNA and DNA. A follow up study visualizes how a structured RNA utilizes these specific ionic signatures to move across the rough folding landscape to a native fold. The second half of the dissertation examines ligand binding RNA interactions through single-molecule kinetic measurements utilizing microfluidic mixing devices. With this approach, we investigate the role of ion and ligand partners in pre-organizing a ligand binding RNA domain and resolve a structural hierarchy related to specific interactions with these elements. Finally, we reveal how key positions in a model RNA sequence modulate ligand binding affinity and suggest RNA design rules for tuning this critical parameter.