Single-probe, high-yield spatial mapping of diverse short RNA motifs

SMOOTHY-FISH enables single-probe visualization of ~20-nucleotide RNA motifs at single-molecule resolution, mapping splice-isoform junctions, individual microRNAs, and RNA conformations in single cells and mouse brain tissue.

• Why it matters: Short RNA motifs were previously spatially invisible because hybridization tiling requires long targets, leaving key RNA features unmeasured in spatial transcriptomics.
• What they did: The authors optimized probe brightness, binding specificity, and target accessibility to reach the practical ceiling for hybridization detection, then mapped short motifs in single cells and mouse brain tissues and validated results with orthogonal methods.
• The result: They found alternatively spliced isoform ratios are tightly regulated across cell populations, subcellular miRNA fractions are precisely maintained, and a designed RNA structure shows a prominent alternative conformation—making short motifs a measurable spatial axis.

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