High-throughput competitive binding assay for targeting RNA tertiary structures with small molecules: application to...

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Identifying small molecules that bind to complex RNA tertiary structures (e.g., pseudoknots, G-quadruplexes) is crucial for developing new therapeutics.
• Existing screening methods often lack the throughput or specificity required for complex RNA targets.

Purpose of the Study:

• To develop and validate a high-throughput competitive binding antisense assay for identifying ligands of RNA tertiary structures.
• To demonstrate the assay's versatility by applying it to both bacterial riboswitches and viral RNA structures.

Main Methods:

• A competitive binding assay was designed where potential ligands compete with a labeled antisense oligonucleotide for binding to a labeled RNA structure.
• The assay was initially customized for the PreQ1-I riboswitch pseudoknot and validated using known ligands and analogues.
• The assay was subsequently adapted for a SARS-CoV-2 G-quadruplex structure and tested with known G-quadruplex ligands.

Main Results:

• The assay successfully identified a hit compound (4494) from a library screen against the Fusobacterium nucleatum riboswitch, which inhibited translation.
• Although the hit's activity was traced to a contaminant, it highlighted the assay's high sensitivity.
• The assay was validated for SARS-CoV-2 G-quadruplex structures, demonstrating its broad applicability.

Conclusions:

• The competitive binding antisense assay is a sensitive and versatile tool for discovering ligands targeting diverse RNA tertiary structures.
• This assay expands the available methodologies for RNA-targeted drug discovery.
• The findings facilitate the development of novel therapeutics by enabling efficient screening for RNA-binding molecules.