Solid-state nanopore sensing reveals conformational changes induced by a mutation in a neuron-specific tRNA<SUP>Arg</SUP>

We demonstrate that solid-state nanopore sensing is a powerful single-molecule method for analyzing RNA conformational ensembles. As a model, we employed n-Tr20, a neuron-specific cytoplasmic tRNA$_{\mathrm{UCU}}<^>{\mathrm{Arg}}$, whose C50U mutation is associated with neurodegeneration in C57BL/6J mice. Maturation of the n-Tr20$<^>{\mathrm{C50U}}$ precursor is impaired as the mutation stabilizes a conformational ensemble different from the wild type. To gain insights into how this mutation engenders structural differences, we used solid-state nanopore sensing for the real-time identification of metastable conformers that are not easily observable by ensemble methods. Ion-current traces recorded using an 8 nm nanopore revealed broad contours of the conformational landscape of n-Tr20/n-Tr20$<^>\mathrm{C50U}$ $\pm$ Mg$<^>{2+}$. Additionally, cryo-electron microscopy analysis and small-angle X-ray scattering studies revealed structural plasticity consistent with the nanopore-sensing data. Since dynamics undergird RNA (dys)function in cellular physiology and pathology, nanopore sensing to determine RNA conformational sampling is a valuable addition to the growing RNA structural analysis toolkit.