Abstract：
This study investigates transfer ribonucleic acid (tRNA) conformational dynamics in the context of MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) using solid-state silicon nitride (SiN) nanopore technology. SiN nanopores in thin membranes with specific dimensions exhibit high signal resolution, enabling real-time and single-molecule electronic detection of tRNA conformational changes. We focus on human mitochondrial tRNALeu(UAA) (mt-Leu(UAA)) that decodes Leu codons UUA/UUG (UUR) during protein synthesis on the mt-ribosome. The single A14G substitution in mt-Leu(UAA) is the major cause of MELAS disease. Measurements of current blockades and dwell times reveal distinct conformational dynamics of the wild-type (WT) and the A14G variant of mt-Leu(UAA) in response to the conserved post-transcriptional m1G9 methylation. While the m1G9-modified WT transcript adopts a more stable structure relative to the unmodified transcript, the m1G9-modified MELAS transcript adopts a less stable structure relative to the unmodified transcript. Notably, these differential features were observed at 0.4 M KCl, but not at 3 M KCl, highlighting the importance of experimental settings that are closer to physiological conditions. This work demonstrates the feasibility of the nanopore platform to discern tRNA molecules that differ by a single-nucleotide substitution or by a single methylation event, providing an important step forward to explore changes in the conformational dynamics of other RNA molecules in human diseases.
摘要：
这项研究调查了MELAS（线粒体脑肌病，乳酸性酸中毒,和中风样发作）使用固态氮化硅（SiN）纳米孔技术。具有特定尺寸的薄膜中的SiN纳米孔表现出高信号分辨率，能够实时和单分子电子检测tRNA构象变化。我们专注于人类线粒体tRNALeu（UAA）（mt-Leu（UAA）），它在mt核糖体上的蛋白质合成过程中解码Leu密码子UUA/UUG（UUR）。mt-Leu(UAA)中的单个A14G取代是MELAS病的主要原因。当前阻断和停留时间的测量揭示了野生型（WT）和mt-Leu（UAA）的A14G变体对保守的转录后m1G9甲基化的反应的不同构象动力学。虽然m1G9修饰的WT转录本相对于未修饰的转录本采用更稳定的结构，m1G9修饰的MELAS转录物采用相对于未修饰的转录物更不稳定的结构。值得注意的是,在0.4MKCl下观察到这些差异特征，但不是在3MKCl，强调更接近生理条件的实验设置的重要性。这项工作证明了纳米孔平台辨别通过单核苷酸取代或通过单个甲基化事件而不同的tRNA分子的可行性。为探索人类疾病中其他RNA分子的构象动力学变化提供了重要的一步。
