Development of Ion Mobility-Mass Spectrometry and Collision-Induced Unfolding for the Structural Characterization of Noncoding Ribonucleic Acids

Abstract

Current small molecules and antibody-based therapies target only a small fraction of the known diseased products of the human genome. While much of the molecular diversity in human cells, particularly non-coding ribonucleic acid (RNA) transcripts, remains unexplored as therapeutic targets, existing therapies have effectively treated various diseases and disorders. Meanwhile, RNA-based biotherapeutics have emerged as a promising new class of treatments. RNA-based biotherapeutics have emerged as a promising new treatment class, with advances in RNA research broadening biotherapeutic possibilities. The success of RNA-based medicines during the 2020 COVID-19 pandemic underscored their potential. However, rigorous characterization is essential to ensure their safety and efficacy. Numerous technologies are currently used for the characterization of RNA higher-order structure (HOS). Over the past two decades, mass spectrometry (MS)-based techniques have been pivotal for protein HOS characterization, but RNA applications have lagged behind. Recent advancements in MS methods targeting RNA analytes are addressing this gap. This dissertation explores extending ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) to RNA HOS characterization. IM-MS enables sorting of RNA mixtures and structures with minimal sample preparation, while CIU rapidly measures RNA stability. This work represents the first application of these structural MS technologies to RNA biomolecules. Chapter 2 details the development of native IM-MS and CIU for RNA HOS characterization. Preliminary experiments with species of varying HOS levels demonstrated CIU’s ability to detect RNA HOS differences under experimentally optimized conditions. While nucleic acid species compact upon desolvation, a key RNA CIU finding was an energy-dependent structural compaction preceding unfolding events. Using a transfer RNA (tRNA) model, renaturation conditions with variable Mg2+ concentrations were studied, and solution-relevant stoichiometries were preserved during desolvation. These conditions were then applied to a disease-related tRNA mutation, uncovering significant structural and stability changes. These findings confirm CIU’s utility for probing disease-related RNA structural perturbations. Chapter 3 investigates lipid nanoparticle (LNP)-induced effects on LNP-encapsulated RNA cargo and is the first MS-directed study for phenomenon. A model LNP system was selected, and with temperature-controlled sample preparation, LNP heterogeneity was minimized. LNP modifications in lipid types and compositions revealed significant RNA structural and stability differences due to RNA-lipid interactions. Weak but notable interactions were identified as key contributors to these effects. Chapter 4 evaluates, for the first time, reagent and electrothermal RNA-supercharging for CIU analysis. These methods generated reproducible and detailed CIU fingerprints that strongly correlated (R2 ≥ 0.95) with solution unfolding measurements across varying Mg2+ concentrations and disease-relevant mutations. This quantitative agreement confirms CIU’s capability of assessing solution-relevant RNA structures. Chapter 5 addresses challenges in assessing RNA secondary higher order structure (HOS) by current RNA CIU conditions and instrumentation workflows. Despite these challenges, energy-dependent analyses of structural transitions by full-width half max (FWHM) provided novel insights not captured by existing IM or CIU methods. Chapter 6 applies the IMgeniusTM benchtop spectrometer to mitochondrial tRNA species from Chapter 2, developed during an internship at IonDX. This study highlights RNA sensitivity to ionization conditions and demonstrates strong alignment between IMgeniusTM data and earlier IM-MS results, confirming the platform's reliability under optimized conditions. The dissertation concludes in Chapter 7 by discussing the future of RNA-focused MS technologies, emphasizing their potential in structural transcriptomics and RNA-based therapeutics. Together, these findings expand the scope of RNA structural biology and provide foundational insights for further innovation in RNA biophysics and therapeutic applications.