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

abcFISH enables robust, high-specificity single-molecule imaging of circular RNAs by targeting back-splicing junction sites, overcoming sequence overlap with linear RNAs.

• Why it matters: Subcellular circRNA localization is key to function, but prior imaging is hindered because circRNA sequences overlap with linear counterparts.
• What they did: The authors built amplicon-based circRNA fluorescence in situ hybridization using optimized rolling-circle amplification at back-splicing junctions for quantitative multiplexed imaging in cells and tissues.
• The result: abcFISH reveals alternative circularization patterns from a single locus, cell-type-specific circRNA expression linked to RNA-binding protein combinatorics, neuron and brain spatial heterogeneity, and colocalization of Cdr1as with Cyrano, plus validation of therapeutic double-stranded circRNA aptamers and reduced astrocyte infiltration around transduced cells.

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