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

Abstract

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 (KD) 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.

1. Introduction

Riboswitches are structured non-coding RNAs that are typically located in the 5′ untranslated regions (UTRs) of mRNAs, where they fold and bind to metabolites or other small molecules to regulate gene expression [1,2]. Riboswitches are actually aptamers that carry expression platforms. Aptamers are single-stranded DNA or RNA molecules that can fold into structures and bind to ligands with high specificity and affinity [3–7]. They have been engineered as various sensitive biosensors and nucleic acid drugs [8–14].

The aptamer and expression platform of riboswitches are two overlapping components [2,15]. The aptamer can bind to ligands, and the expression platform can turn on or off gene expression. Examples of expression platforms include ribosomal binding sites (RBSs), rho-independent terminators, and splicing site proximal regions [1,2,16–18]. The conformation of the aptamer changes upon ligand binding, resulting in exposure or sequestration of the expression platform. In addition, tandem riboswitches, such as the guanine aptamer combined with the phosphoribosyl pyrophosphate (PRPP) aptamer, can function as an IMPLY Boolean logic gate to regulate the transcription of messenger RNAs for purine biosynthesis in bacteria [19], indicating their possible applications as logic control circuits [19,20]. Hence, these structured RNAs serve as genetic switches that can activate or repress gene expression in response to changes in the levels of their target ligands [1,17,21].