Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Department of Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
J Am Chem Soc. 2024 Feb 21;146(7):4412-4420. doi: 10.1021/jacs.3c09230. Epub 2024 Feb 8.
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 study, we describe the development of native ion mobility-mass spectrometry 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 in 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-associated A3243G mutation. Our data demonstrate that our experimentally determined conditions preserve some solution-state memory of RNAs via the correlated complexity of CIU fingerprints and RNA HOS, the observation of predicted stability shifts in the control RNA samples, and the retention of predicted magnesium binding events in gas-phase RNA ions. Significant differences in collision cross section and stability are observed as a function of the A3243G mutation across a subset of the mitochondrial tRNA maturation pathway. We conclude by discussing the potential application of CIU for the development of RNA-based biotherapeutics and, more broadly, transcriptomic characterization.
核糖核酸（RNAs）仍然是结构生物学的挑战性目标，这为理解它们在细胞生物学中的广泛功能以及充分实现其在生物技术中的应用造成了障碍。RNAs 的固有动态性在捕捉其具有生物学相关性的高级结构（HOS）方面带来了诸多障碍，因此，许多 RNA 功能仍然未知。在这项研究中，我们描述了用于各种 RNA 的结构表征的天然离子淌度-质谱和碰撞诱导解折叠（CIU）的开发。我们评估了这些技术在跨越多种功能 RNA 时在气相中保留天然结构特征的能力。最后，我们应用这些工具来研究难以捉摸的线粒体脑病、乳酸酸中毒和中风样发作相关的 A3243G 突变。我们的数据表明，我们通过 CIU 指纹图谱和 RNA HOS 的相关性复杂性、对照 RNA 样品中预测稳定性变化的观察以及预测镁结合事件在气相 RNA 离子中的保留，证明了我们实验确定的条件通过保留一些溶液状态记忆来保留 RNA。在一小部分线粒体 tRNA 成熟途径中，观察到作为 A3243G 突变函数的碰撞截面和稳定性的显著差异。最后，我们讨论了 CIU 在 RNA 为基础的生物治疗剂的开发以及更广泛的转录组表征方面的潜在应用。
