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bioRxiv · 10.1101/2025.11.16.688740

abcFISH enables multiplexed, single-molecule visualization of circular RNA spatial heterogeneity

Liu, Y.-X.·Jiang, B.-W.·Liu, K.-M.·Yang, L.·Chen, L.-L.
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Abstract

Functional RNAs often exhibit distinct subcellular localization, but studying circular RNA (circRNA) localization has been challenging due to their extensive sequence overlap with linear mRNAs. We developed amplicon-based circular RNA fluorescence in situ hybridization (abcFISH), a method that employs an optimized rolling circle amplification (RCA) strategy targeting back-splicing junction (BSJ) sites for robust, high-specificity, single-molecule circRNA imaging. abcFISH enables quantitative, multiplexed imaging in cells and tissues, allowing us to reveal alternative circularization patterns from the single locus; uncover cell-type-specific circPOLR2A(9,10) expression related to a combinatorial effect of RNA-binding proteins; map the distinct spatial distribution patterns of multiple circRNAs in neurons and brain tissues; and show the functional interplay of circRNA Cdr1as and lncRNA Cyrano co-localization. Furthermore, we applied abcFISH to validate the efficacy of therapeutic double-stranded circRNA aptamers (ds-cRNAs), simultaneously visualizing AAV-delivered ds-cRNAs and a consequent reduction in astrocyte infiltration around transduced cells. Collectively, abcFISH establishes a robust and user-friendly toolkit for deciphering circRNA localization and function in vivo.

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Liu, Y.-X., Jiang, B.-W., Liu, K.-M., Yang, L., Chen, L.-L.. 2025-11-17. abcFISH enables multiplexed, single-molecule visualization of circular RNA spatial heterogeneity. https://doi.org/10.1101/2025.11.16.688740

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