Rearrangement of Partially Ordered Stacked Conformations Contributes to the Rugged Energy Landscape of a Small RNA Hairpin

We have studied the fast relaxation kinetics of a small RNA hairpin tetraloop using time-resolved infrared spectroscopy. A laser-induced temperature jump initiated the relaxation by rapidly perturbing the thermal equilibrium of the sample. We probed the relaxation kinetics at two different wavenumbers, 1574 and 1669 cm⁻¹. The latter is due to the C6O6 carbonyl stretch of the base guanine and is a direct measure of guanine base pairing. The former is assigned to a ring vibration of guanine and tracks structure by sensing base stacking interactions. Overall, the kinetics at 1574 cm⁻¹ are faster than those observed at 1669 cm⁻¹. When relaxation occurs at the melting temperature, the kinetics at both wavenumbers are biexponential. When relaxation occurs at a temperature that is higher than the melting temperature, the data at 1669 cm⁻¹ are still biexponential while only a single fast phase is resolved in the data at 1574 cm⁻¹. The fast phases are in the range of microseconds, while the slower phases are in the range of tens of microseconds. At both wavenumbers, a portion of the relaxation is not resolved, indicating the existence of a very fast, sub-100 ns phase. Our results provide additional evidence that small, fast folding hairpin loops are characterized by a rugged energy landscape. Furthermore, our data suggest that single-strand stacking interactions and stacking interactions in the loop contribute significantly to the ruggedness of the energy landscape. This work also demonstrates the utility of time-resolved infrared spectroscopy in studying RNA folding.