Quantification of disease-associated RNA tandem repeats by nanopore sensing

Patino-Guillen, G.; Pesovic, J.; Panic, M.; Earle, M.; Ninkovic, A.; Petrusca, S.; Savic-Pavicevic, D.; Keyser, U. F.; Boskovic, F.

doi:10.1101/2025.05.15.653973

bioRxiv · 10.1101/2025.05.15.653973

Quantification of disease-associated RNA tandem repeats by nanopore sensing

Patino-Guillen, G.·Pesovic, J.·Panic, M.·Earle, M.·Ninkovic, A.·Petrusca, S.·Savic-Pavicevic, D.·Keyser, U. F.·Boskovic, F.

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Abstract

Short tandem repeat expansions underlie a class of neurological and neuromuscular diseases known as repeat expansion disorders (REDs), yet the precise characterization of these repeats remains technically challenging. Conventional amplification-based methods fail to resolve repeat length accurately due to amplification bias and sequence homogeneity. Here, we present a single-molecule nanopore-based strategy that enables direct quantification of tandem repeats in native RNA. By assembling RNA:DNA nanostructures that encode specific repeat number, we achieve repeat size discrimination with a resolution of 18 nucleotides. Using tandem repeat-containing RNA, we successfully detect and discriminate disease-relevant repeat lengths associated with myotonic dystrophy types 1 (DM1) and 2 (DM2), and congenital central hypoventilation syndrome-1 (CCHS1). Finally, we apply our method to total RNA extracted from a DM1 human cell line model, demonstrating its compatibility with complex biological samples. Our approach offers a platform for studying repeat expansion biology at the single-molecule level, with broad implications for diagnostics, clinical research and multiplexed repeat profiling.

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Patino-Guillen, G., Pesovic, J., Panic, M., Earle, M., Ninkovic, A., Petrusca, S., Savic-Pavicevic, D., Keyser, U. F., Boskovic, F.. 2025-05-16. Quantification of disease-associated RNA tandem repeats by nanopore sensing. https://doi.org/10.1101/2025.05.15.653973

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