High-throughput competitive binding assay for targeting RNA tertiary structures with small molecules: application to pseudoknots and G-quadruplexes

There is an indisputable need for new screening methodologies to identify small molecules that target RNA tertiary structures, such as pseudoknots or G-quadruplexes. Here, we present a high-throughput competitive binding antisense assay designed to identify ligands for complex RNA tertiary structures. In this assay, initially customized for the bacterial PreQ1-I riboswitch pseudoknot, ligands compete with a rationally designed quencher-labelled antisense oligonucleotide for binding to a fluorophore-labelled riboswitch. The method is validated for four PreQ1-I riboswitches, using the natural riboswitch ligand PreQ1 and various analogues. A commercial RNA-focused library consisting of ∼15 000 compounds was then screened against the Fusobacterium nucleatum riboswitch, leading to the identification of a promising hit, 4494, which showed competitive binding activity to all PreQ1-I riboswitches and was able to inhibit translation of a riboswitch-regulated reporter gene. Although resynthesis of 4494 revealed that its activity originated from an ∼1% guanine contamination, this result underscores the assay's exceptional sensitivity. To demonstrate its versatility, the assay was tailored for a SARS-CoV-2 G-quadruplex structure and validated with several known G-quadruplex ligands. This work shows that the competitive binding antisense assay is a powerful addition to the RNA-targeting toolbox, facilitating the discovery of ligands for diverse RNA tertiary structures.