A competitive binding assay for RNA ligand discovery

Wintermans, S.E.L. (2026)

Doctoral Thesis

In the emerging field of small-molecule RNA targeting, there is a growing demand for new high-throughput screening methods. This thesis describes the development of a competitive binding antisense assay (CB ASsay), in which small molecules compete with a specifically designed antisense oligonucleotide to bind to the target RNA. This allows for the identification of new RNA ligands for clinically relevant RNA tertiary structures. The CB ASsay was employed to three pathogenic RNA targets: the bacterial PreQ₁ riboswitch, the SARS-CoV-2 frameshifting pseudoknot and the G-quadruplex at position 3467 in the SARS-CoV-2 genome (GQ-3467). The CB ASsay is highly sensitive, applicable to various RNA structures and is particularly well-suited for high-throughput screenings. Screening the Enamine RNA-focused library and the European Lead Factory library led to the identification of several hit molecules that can now be investigated for their antimicrobial properties. All in all, this thesis demonstrates that the CB ASsay is an important addition to the RNA-targeting toolbox.

• RNA
• RNA-targeting
• PreQ1 riboswitch
• SARS-CoV-2
• Pseudoknot
• G-quadruplex
• High-throughput screening
• Small molecules
• Antibacterial
• Antiviral

• All authors: Wintermans, S.E.L.
• Supervisor: Aerts, J.M.F.G.
• Co-supervisor: Olsthoorn, R.C.L.; Artola Perez de Azanza, M.E.
• Committee: Ubbink, M.; Stelt, M. van der; Dame, R.T.; Rentmeister, A.; Geurink, P.P.; Velema, W.A.
• Qualification: Doctor (dr.)
• Awarding Institution: Leiden Institute of Chemistry (LIC), Faculty of Science, Leiden University
• Date: 2026-02-24
• ISBN (print): 9789465370521
• Dataset: https://doi.org/10.5281/zenodo.10912410