• • Optimal cleavage of the integrated hammerhead ribozyme requires a bridge length of 3–6 base pairs; deviations reduce cleavage efficiency, directly impacting the sensitivity and dynamic range of ligand-binding detection in biosensor and gene-control applications.
• • The method achieves KD values concordant with in-line probing, using only 1 pmol of allosteric ribozyme RNA per measurement, which reduces material consumption by orders of magnitude compared to conventional techniques and enables high-throughput screening of riboswitch candidates.
• • Riboswitch candidate Motif_9307 exhibits no binding affinity for S-adenosylmethionine or several other tested ligands, as confirmed by both the ribozyme-based assay and in-line probing, validating the method's specificity and reliability for negative screening.
• • Addition of yeast extract as a complex ligand mixture increases cleavage activity, indicating the presence of the Motif_9307 ligand in the extract; this demonstrates the assay's capacity to detect unknown ligands in complex biological matrices, with implications for natural product discovery and metabolic pathway engineering.