Structural Characterization of Ribonucleic Acids and Their Complexes by Negative-ion Mode Mass Spectrometry

Abstract

Ribonucleic acids (RNAs) form complexes with deoxyribonucleic acids, proteins, other RNAs, and smaller ligands. Detailed knowledge of RNA interaction sites provides a basis for understanding functions. With limited analytical techniques available to obtain deeper understanding of RNA structure, negative ion mode mass spectrometry (MS) has the potential to significantly expand RNA primary, secondary, tertiary, and quarternary structure information. This dissertation presents novel MS methods for characterizing RNAs and their complexes. Negative-ion electron capture dissociation (niECD) involves ~3.5-6.5 eV electron irradiation to yield charge-increased intermediates that further undergo radical-driven fragmentation. The proposed niECD mechanism involves gas-phase zwitterionic structures in which nucleobases are protonated and the phosphate backbone is deprotonated. We found that electron-capture efficiency is higher for purine nucleobases compared with pyrimidines and that purine radicals are more stable, presumably because purines have higher proton affinities and can form intramolecular hydrogen bonds. niECD efficiency decreases with increased charge state due to Coulomb repulsion. We show that gas-phase proton-transfer reactions can be combined with niECD for improved performance. Electrospray ionization (ESI) of a model RNA hairpin from native-like (10 mM ammonium acetate) and methanol-containing (up to 50%) solvents resulted in identical charge state distributions, suggesting a minor methanol effect on overall conformation. Experimentally determined collision cross sections (CCSs) for the 5- and 6- charge states of this RNA are smaller (789 Å2 and 830Å2, respectively) than those predicted from the NMR structure. Replica-exchange molecular dynamics showed that these charge states adopt globular collapsed structures due to self-solvation whereas the 7- charge state showed hairpin retention. Higher charge states showed extended structures (higher CCSs). Ligand (e.g., paromomycin) binding assays at varied methanol content resulted in strongest binding at 0% methanol (64+6 nM KD). However the KD remained within one standard deviation up to 50% methanol, suggesting that the binding site is mainly unperturbed in methanol. Assays at varied pHs showed strongest binding at neutral pH. Overall, these data suggest that moderate methanol concentrations, which facilitate ESI, can be tolerated in native RNA MS. Crosslinking techniques coupled with MS provide an alternative tool for identifying RNA interaction sites. We show that collisional activation can provide full sequence coverage of the RNA moiety within non-covalent RNA-peptide complexes; however complexes are disrupted, resulting in loss of site-specific information. By contrast, niECD, in combination with infrared multiphoton dissociation provided sufficient sequence coverage while retaining non-covalent interactions. We also show that IR irradiation at 10.6 µm selectively dissociates RNA-peptide crosslinked species within a peptide mixture due to resonance absorption by phosphate groups, thus allowing identification of such species. Microfluidics is a highly efficient technology for biological analysis. Microfluidic-type approaches, including nano-ESI and nano-LC, coupled with MS provide several advantages, e.g., limited sample consumption and enhanced sensitivity. In order to disseminate microfluidic principles, we developed a 2-week (8 hour) laboratory experiment for an undergraduate analytical chemistry course. Students are introduced to soft lithography concepts by designing/characterizing their own agar-based microfluidic chips. They learn about fluid dynamics by approaching the challenge of mixing in microfluidic channels. By varying solvent viscosity and channel geometries, terms that govern the Reynolds number, students achieve mixing. The optimal chip geometry/solvent condition is used to quantify salicylic acid-/iron (III) complex by colorimetric analysis. Overall, this dissertation describes the utility of MS (and its associated tools) for the study of RNA, RNA-small molecule, and RNA-protein complexes.