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bioRxiv · 10.1101/2025.04.08.647894

Solid-state nanopore sensing reveals conformational changes induced by a mutation in a neuron-specific tRNAArg

Dutt, S.·Lai, L. B.·Mehta, R.·Karawdeniya, B. I.·Bandara, Y. M. N. D. Y.·Clulow, A. J.·Glatt, S.·Gopalan, V.·Kluth, P.
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Abstract

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 tRNAArgUCU, whose C50U mutation is associated with neurodegeneration in C57BL/6J mice. Maturation of the Tr20C50U 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-Tr20C50U {+/-} Mg2+. Additionally, cryo-EM analysis and small-angle X-ray scattering studies revealed structural plasticity even more than predicted from 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/647894v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@45bforg.highwire.dtl.DTLVardef@1e31110org.highwire.dtl.DTLVardef@75388forg.highwire.dtl.DTLVardef@1d45037_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Dutt, S., Lai, L. B., Mehta, R., Karawdeniya, B. I., Bandara, Y. M. N. D. Y., Clulow, A. J., Glatt, S., Gopalan, V., Kluth, P.. 2025-04-09. Solid-state nanopore sensing reveals conformational changes induced by a mutation in a neuron-specific tRNAArg. https://doi.org/10.1101/2025.04.08.647894

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