Detailed Kinetics Of Rna Folding Pathways And Thermodynamic Origins Of Crowding By Single-Molecule Fret

Specific three dimensional folding is required for RNA function while RNA can be a highly dynamic system that actively samples different configurations under thermal fluctuations. For instance, in gene regulation of RNA riboswitch, the prompt structural transition in response to ligand is crucial for triggering transcriptional termination, making the time scale of folding and ligand response exceedingly relevant to RNA functions. In single-molecule FRET (smFRET) experiments, the conformational changes of RNA are visualized by the time-dependent energy transfer to determine folding and unfolding rates. Such kinetic information allows us to construct a model for manganese riboswitch folding and explore the detailed folding mechanisms, where Mg2+ binding is found to pre-fold the RNA, followed by ligand (Mn2+) association to further stabilize the folded structure. Moreover, the thermodynamic origins can be obtained from temperature dependent folding. The manganese riboswitch folding is found unexpectedly promoted by heat and the process is endothermic with net entropy gain, much contradictory to the conventional idea of folding into a more order state. In molecular crowding study where we try to understand the influence of fundamental excluded volume on RNA structures in highly concentrated intracellular environments, the chemically inert crowders are shown to entropically favor the more compact folded structure of RNA; a simple physical picture of osmotic pressure is employed to explain the crowder size dependence. Lastly, a hydraulic pressure generator is used to apply pressure (up to 5 kbar) to RNA sample through capillary. The free volume change (pΔV) during RNA folding may be related to structural response under crowding conditions.