Ribonucleic acids (RNAs) are becoming increasingly important to our understanding of cellular biology and our search for new or improved biotherapeutics. However, the dynamic and polydisperse nature of RNAs creates challenges for their structural characterization. Here, we focus on RNA stem-loops (i.e., hairpins), as these are ubiquitous elements of RNA secondary structures. We leverage native ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) to study stem loops isolated from mitochondrial tRNA (tRNA) across a wide range of disease-relevant mutants. While we find that native IM-MS analyses proved capable of tracking stem-loop structure changes upon cofactor-binding and mutation, it remains challenging to evaluate the stabilities of small RNAs using standard RNA CIU workflows. Thus, we explore the ability of energy-dependent changes by IM peak full-width half-maximum (fwhm) values to characterize the stabilities of RNA stem-loops. We find that RNA stem-loop IM fwhm values typically decrease significantly upon activation, with lesser differences detected in Mg2+-bound RNA samples in a manner consistent with the stabilization expected under such conditions. We conclude by discussing the potential future applications of IM fwhm analyses in the context of RNA CIU assay development.

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