Investigating the thermodynamics of UNCG tetraloops using infrared spectroscopy

Using infrared (IR) absorption spectroscopy, we have explored the folding thermodynamics of the UNCG class of RNA hairpin tetraloops (N = U, A, C, or G). Without the need to introduce non-native probes, IR spectroscopy makes it possible to distinguish specific structural elements such as base pairing versus base stacking or loop versus stem motions. Our results show that different structural components exhibit different thermodynamics. Specifically, we have found that tetraloop melting proceeds in a thermally sequential fashion, where base pairing in the stem is disrupted before (i.e., at a lower temperature) base stacking along the entire chain. In addition, for N = A, our data argue that the structure immediately surrounding adenine is particularly stable and melts at a higher temperature than either base-pairing or base-stacking interactions. Taken together, these results suggest that hairpin loop formation is not a simple two-state process, even in the equilibrium limit.