Exploring the effects of temperature and pressure on the structure and stability of a small RNA hairpin.

RNAs perform multiple vital roles within cells, including catalyzing biological reactions and expression of proteins. Small RNA hairpins (sRNAh) are the smallest functional entities of nucleic acids and are involved in various important biological functions such as ligand binding and tertiary folding initiation of proteins. We investigated the conformational and free energy landscape of the sRNAh gcUUCGgc over a wide range of temperatures and pressures using fluorescence resonance energy transfer, Fourier-transform infrared and UV/Vis spectroscopy as well as small-angle X-ray scattering on the unlabeled and/or fluorescently labeled sRNAh. The sRNAh shows a broad melting profile with continuous increase of unpaired conformations up to about 60°C. However, the sRNAh structure might not be fully unfolded at temperatures as high as 90°C and still comprise various partially unfolded compact conformations. Pressure up to 400MPa has a small effect on the base pairing and base stacking interactions of the sRNAh, indicating small conformational perturbations, only, which might originate from minor changes in packing and hydration of the RNA molecule upon compression. Pressurization at 70°C, i.e. at a temperature above the melting transition, has no significant effect on the conformational ensemble of the sRNAh, i.e., it does not promote formation of new native stem connections after thermal denaturation. Finally, we noticed that Cy3/Cy5 labeling of the sRNAh changes, probably via stacking interactions between the fluorescent dyes and the nucleotide rings, the stability of the sRNAh, thereby rendering FRET analysis of the conformational dynamics of such small RNA structure inappropriate.