Collision Induced Unfolding Reveals Disease-Associated Stability Shifts in Mitochondrial tRNAs

Ribonucleic acids (RNAs) remain challenging targets for structural biology, creating barriers to understanding their vast functions in cellular biology and fully realizing their applications in biotechnology. The inherent dynamism of RNAs creates numerous obstacles in capturing their biologically relevant higher-order structures (HOSs), and as a result, many RNA functions remain unknown. In this report, we describe the development of native ion mobility-mass spectrometry (IM-MS) and collision induced unfolding (CIU) for the structural characterization of a variety of RNAs. We evaluate the ability of these techniques to preserve native structural features into the gas-phase across a wide range of functional RNAs. Finally, we apply these tools to study the elusive mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions preserve some solution-state memory of RNA via the increasing complexity of CIU fingerprints by RNA HOS and the retention of predicted magnesium binding events. Relating to the role of MELAS-inducing mutations in mitochondrial trans-fer RNA (mt-tRNAs), significant differences in collision cross-section (CCS) and stability are observed as a function of the A3243G mutation across a subset of the mitochondrial transfer RNA (tRNA) maturation pathway. We conclude by discussing the potential application of CIU for the development of RNA-based biotherapeutics and, more broadly, transcriptomic characterization.