Integration of the hammerhead ribozyme into structured RNAs to measure ligand-binding events for riboswitch candidates and aptamers

• A novel method integrates a hammerhead ribozyme into structured RNAs via a multibase pair bridge to detect ligand binding, with optimal cleavage achieved at 3-6 base pairs. • The method yields dissociation constants consistent with in-line probing and requires only 1 pmol of RNA, offering high sensitivity. • Application to riboswitch candidate Motif_9307 showed no binding to SAM or other tested ligands, but detected a ligand in yeast extract, demonstrating its utility in complex mixtures. • This approach provides a convenient alternative for measuring ligand-binding events, facilitating the characterization of riboswitch candidates and aptamers.