High-throughput competitive binding assay for targeting RNA with small molecules: discovery of new PreQ₁riboswitch ligands

Abstract

In the evolving landscape of RNA targeting, there is an indisputable need for new screening methodologies to find small molecules targeting relevant tertiary RNA structures, like viral pseudoknots or bacterial riboswitches. Here, we developed a competitive binding high-throughput screening assay to identify ligands for the bacterial PreQ1-I riboswitch. In this assay, ligands compete with a rationally designed quencher-labeled antisense for binding to the riboswitch. The method is validated for the Fusobacterium nucleatum (Fnu), Thermoanaerobacter tengcongensis (Tte), Bacillus subtilis (Bsu) and Enterococcus faecalis (Efa) PreQ1 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 Fnu riboswitch, leading to the identification of 4 hits exhibiting competitive binding activity. These hits were evaluated in in vitro translation assays against several PreQ1 riboswitches. The most promising hit 4494 showed competitive binding activity to the Fnu, Tte, Bsu and Efa riboswitches, and was able to inhibit translation of a Tte riboswitch-regulated reporter gene, making it an interesting starting point for the development of new antibiotics. In essence, this HTS assay has the potential to discover highly sought-after RNA targeting small molecules for complex and clinically relevant RNA structures.