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

Nucleic Acids Research, vol. 53

Abstract

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.

Authors 12

1. Sophie E.L. Wintermans Aachen Department of Medical Biochemistry

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

   Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

2. Jana S Hoffmann Aachen Department of Medical Biochemistry

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

   Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

3. Victor van Kuijk Aachen Department of Medical Biochemistry

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

   Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

4. Yelda Durmus Aachen Department of Medical Biochemistry

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

   Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

5. Mariska D. Tacoma Aachen Department of Medical Biochemistry

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

   Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

6. Indy Broekhuizen Aachen Department of Medical Biochemistry

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

   Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

7. Antonius P. A. Janssen Aachen

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Molecular Physiology, , Einsteinweg 55, 2333 CC Leiden,

8. Hans van den Elst Aachen Department of Bio-organic Synthesis

   Leiden University

   Affiliation as printed

   Leiden Institute of Chemistry, Leiden University Department of Bio-organic Synthesis, , Einsteinweg 55, 2333 CC Leiden,

   Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

9. Bjorn R. van Doodewaerd Aachen Division of Chemical Biology and Drug Discovery

   Leiden University Medical Center

   Affiliation as printed

   Division of Chemical Biology and Drug Discovery, Leiden University Medical Center Department of Cell and Chemical Biology, , Einthovenweg 20, 2333 ZC Leiden,

10. Paul P. Geurink Aachen Division of Chemical Biology and Drug Discovery

    Leiden University Medical Center

    Affiliation as printed

    Division of Chemical Biology and Drug Discovery, Leiden University Medical Center Department of Cell and Chemical Biology, , Einthovenweg 20, 2333 ZC Leiden,

11. Marta Artola corresponding Aachen Department of Medical Biochemistry

    Leiden University

    Affiliation as printed

    Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

    Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands

12. René C. L. Olsthoorn corresponding Aachen Department of Medical Biochemistry

    Leiden University

    Affiliation as printed

    Leiden Institute of Chemistry, Leiden University Department of Medical Biochemistry, , Einsteinweg 55, 2333 CC Leiden ,

    Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands