Rolling Circle Amplification With Hairpin Probes for In Situ Single‐Molecule RNA Imaging at Single‐Base Resolution

We present an in situ single-molecule RNA imaging method, termed hairpin-primed rolling circle amplification (RCA) imaging assay (HPRIA), which achieves single-base resolution and subcellular localization. This method utilizes the metastable stem-loop structure of hairpin probes, which unfolds only when perfectly complementary to the target RNA, thereby enabling the imaging of RNA molecules with single-nucleotide polymorphism (SNP) discrimination and single-base precision. By integrating T4 DNA ligase-mediated padlock probe circularization with Phi29 DNA polymerase-based RCA, HPRIA enables highly efficient and specific signal amplification from individual RNA molecules. We demonstrate the method's applicability to in situ single-molecule imaging of both mRNA and miRNA, as well as for spatial localization of multiple genes at subcellular resolution. HPRIA thus provides a powerful and versatile tool for studying cellular heterogeneity and RNA-related disease mechanisms.