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

Spatial transcriptomics has revolutionized our understanding of RNA biology. Hybridization-based methods map mRNA transcripts by tiling multiple labeled probes along each molecule to generate a visible fluorescent signal, restricting applicability to long transcripts. Short RNA motifs, consequently, can only accommodate a single probe and remain a spatially invisible dimension of RNA biology. Here we present SMOOTHY-FISH, a single probe strategy that directly visualizes short RNA motifs at the single-molecule resolution. By optimizing probe brightness, binding specificity, and target accessibility, our single ~20-nucleotide probe reached the practical ceiling of hybridization-based detection. We quantitatively mapped previously inaccessible short motifs - splice-isoform junctions, individual microRNAs, and structural conformations - in single cells and mouse brain tissues, each validated against orthogonal methods. This unveiled biological phenomena that were previously unknown: ratios of alternatively spliced isoforms are tightly regulated across cell populations, subcellular miRNA fractions are precisely maintained, and a designed RNA structure was found to have a prominent alternative conformation. These findings demonstrate that short RNA motifs possess key spatial information that has yet to be discovered. With SMOOTHY-FISH's optimized single probe technique, short RNA motifs are converted into a new routinely measurable axis of spatial transcriptomics.