To understand the influence of secondary structure on RNA folding and unfolding, it is essential to investigate the kinetics of hairpin loop formation, base-pairing, and base-pair disruption in RNA stem-loop hairpin structures. In the past, several studies suggest hairpin folding is not always a two state process, but can proceed by way of on- or off-pathway intermediates. Here we combine both stopped-flow kinetics and fluorescence correlation spectroscopy (FCS) techniques to probe the folding/unfolding kinetics of several RNA hairpins with the same base pair stem sequence and different loop sequences. Each hairpin is labeled with fluorophore (TAMRA) and quencher (DABCYL) on the 3' and 5' end respectively. These measurements are performed over various ranges of KCl concentrations to carefully monitor the formation and disruption of the stem by fluorescence. Stopped-flow kinetics technique is a rapid mixing technique to measure the reaction kinetics of hairpin folding on the milliseconds time scale; whereas, FCS is a single molecule technique to elucidate reaction kinetics in the microseconds time scale. Specially designed RNA molecules exhibit fluorescent fluctuations upon adopting certain secondary structures. FCS monitors the fluorescence of labeled RNA molecules flowing through two laser probe regions. FCS can simultaneously measure different parameters such as diffusion constant, flow velocity, and reaction time constant. Our stopped-flow kinetics and FCS studies show that RNA hairpin folding and unfolding reactions occur over a broad range of time scales from the microseconds to milliseconds range. This suggests the involvement of intermediate folded structures with varying stabilities in the mechanism of RNA hairpin formation and disruption.

Investigation of the kinetics of hairpin loop formation, base pairing and base pair disruptions in RNA hairpin folding/unfolding

242nd National Meeting (2011)

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