Single-Molecule Study of RNA Hairpin and Pseudoknot Folding

Understanding the folding mechanism of RNAs is critical for studying their structure-function relationship. The folding energy landscape of RNA is often rugged, involving multiple folding routes and intermediates. Optical tweezers is a powerful tool to study the folding of RNA, as it enables direct investigation of single RNA molecules and their folding pathways. In this dissertation, I used optical tweezers to investigate the folding of two RNA structural motifs, a hairpin and a pseudoknot. RNA hairpin, the A-form double helix capped with a terminal loop, is a basic RNA secondary structure. I examined a model RNA hairpin, P5ab, derived from the group I intron ribozyme. P5ab was only considered as a two-state folder in previous single-molecule experiments. Here, I have identified multiple intermediate states during the folding of P5ab. These intermediate states are located near the bulge, internal loop, and terminal loop-closing regions. The folding free energy of P5ab was computed from both equilibrium unfolding/folding kinetics and non-equilibrium work done using fluctuation theorems. The features of the energy barrier were explored by model-dependent fit of the unfolding force and kinetics, as well as by equilibrium sampling of the hopping transitions near the equilibrium force. Pseudoknot is an RNA tertiary structure that contains base pairing between the hairpin loop and an unpaired region in the RNA. Here, I examined the class I preQ1 riboswitch aptamer, which folds into a pseudoknot upon binding to the ligand preQ1. Using mechanical unfolding and mutational studies, I elucidated the folding pathways of the aptamer. In the absence of ligand, only a hairpin state is observed, while in the presence of preQ1, the pseudoknot is formed through the hairpin intermediate, and shows a ligand concentration-dependent folding kinetics. This study also serves as the basis for future investigation of the riboswitch control mechanism involving both the aptamer and the expression platform.