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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.

Ultrathin nanopore sensors allow single-molecule and polymer measurements at sub-microsecond time resolution enabled by high current signals (∼10–30 nA). We demonstrate for the first time the experimental probing of the ultrafast translocation and folded dynamics of double-stranded DNA (dsDNA) through a nanopore at 10 MHz bandwidth with acquisition of data points per 25 ns (150 MB/s). By introducing a rigorous algorithm, we are able to accurately identify each current level present within translocation events and elucidate the dynamic folded and unfolded behaviors. The remarkable sensitivity of this system reveals distortions of short-lived folded states at a lower bandwidth. This work revisits probing of dsDNA as a model polymer and develops broadly applicable methods. The combined improvements in sensor signals, instrumentation, and large data analysis methods uncover biomolecular dynamics at unprecedentedly small time scales.