Probing non-coding RNA structural dynamics with 2-aminopurine

This thesis investigates sequence-dependent structural dynamics of non-coding (nc) RNAs utilizing the fluorescent base 2-Aminopurine (2Ap). We conclude that the highly homologous adenine (ARNA) and guanine (GRNA) riboswitches exhibit distinct structural dynamics in the ligand-bound state. Relative to ARNA, GRNA is more preorganized towards the closed/native conformer (CC), displays enhanced thermostability, and higher magnesium (Mg2+) binding affinity. We then focus on the role of nucleoporin 50-kilodalton (Nup50) Alu double-stranded (ds) RNAs in adenosine-to-inosine (A-to-I) editing. Here, we deduce the folding pathways of three constructs containing an internally substituted 2Ap, which are representative of Nup50-Alu dsRNAs in human, chimp, and rhesus species, respectively. Our fluorescence-based data do not fit well to a two state (folded/unfolded) model, but are well modeled by a four-state (two intermediate) model. Despite the high interspecies homology, the human sequence is most heavily edited in vivo. Interestingly, we find that the 2Ap at position five in the human Nup50-Alu dsRNA has the most thermodynamically stable intermediate, which is an optimal substrate for the A-to-I editing enzyme. The investigations of ncRNA folding dynamics underscore how Nature utilizes subtle sequence variations to achieve remarkable diversity.