Unraveling RNA Conformation Dynamics in Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episode Syndrome with Solid-State Nanopores
Srilahari Namani, Kyril Kavetsky, Chih‐Yuan Lin, Sunita Maharjan, Howard Gamper, Nan‐Sheng Li, Joseph A. Piccirilli, Ya‐Ming Hou, Marija Drndić
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 m 1 G9 methylation. While the m 1 G9-modified WT transcript adopts a more stable structure relative to the unmodified transcript, the m 1 G9-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.