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

Some structured RNAs, such as riboswitches and aptamers, can bind to their cognate ligands and have been used in biosensors and gene expression control elements. However, current methods for detecting ligand binding to structured RNAs are either severely limited or inconvenient. In this study, we design a multibase pair bridge to integrate a hammerhead ribozyme into structured RNAs to detect ligand binding events. The experimental results demonstrate that the length of the bridge has a significant effect on the cleavage of the ribozyme; optimal cleavage can be achieved with three to six base pairs in the bridge. The dissociation constant ( <i>K</i> _D) values obtained through this method are in agreement with those determined by in-line probing techniques, and 1 pmol of allosteric ribozyme RNA is sufficient for measurement. We apply this method to evaluate the binding affinity of the riboswitch candidate Motif_9307. Our findings indicate that this motif has no binding affinity for S-adenosylmethionine or several other tested ligands, which is consistent with the results of the in-line probing experiments. Notably, our method reveals an increase in cleavage activity when yeast extract is added as a mixture of ligands, suggesting that the ligand of Motif_9307 is present in the extract. In conclusion, we develop an alternative approach for measuring ligand binding events associated with riboswitch candidates and aptamers.