Biochemical Characterization of a Non-G4-Type RNA Aptamer That Lights Up a GFP-like Fluorogenic Ligand

The 17-3 RNA aptamer recognizes DMHBI and induces its fluorescence. We showed that the 17-3 RNA aptamer predominantly induced emission of the phenolate form of DMHBI. We also demonstrated that the active structure of the minimal form of the 17-3 aptamer possessed three stem elements and two large loop elements, which we named Karashi and its sequence-optimized variant, Jigarashi, respectively. Chemical modification experiments suggested that the two loop regions formed tertiary interactions and/or non-Watson-Crick base pairs, and no remarkable structural alterations occurred upon DMHBI binding. AlphaFold3 also predicted a tertiary structure of the ligand-free form of Jigarashi RNA, which was consistent with the results of chemical modification experiments.