Disulfide Tethering to Map Small Molecule Binding Sites Transcriptome-wide.

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Understanding RNA-small molecule interactions is crucial for drug discovery and development.
• Existing methods may not fully capture the complexity of these interactions across the transcriptome.

Purpose of the Study:

• To develop and validate a novel transcriptome-wide screening method, Tether-seq, for identifying RNA-small molecule interactions.
• To investigate the binding sites of risdiplam, an RNA-targeting drug for spinal muscular atrophy (SMA).

Main Methods:

• Tether-seq utilizes s⁴U metabolic labeling for reversible and covalent attachment of small molecule disulfides to RNA.
• Screening under reducing conditions differentiates binding-stabilized interactions from RNA reactivity.
• SHAPE-MaP (Selective 2′-Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling) was used for structure probing.

Main Results:

• Tether-seq successfully identified potential RNA-small molecule binding sites across the transcriptome.
• A disulfide analogue of risdiplam showed prominent binding to a site within the cytochrome C oxidase 1 (COX1) transcript.
• SHAPE-MaP confirmed a structured motif and binding of risdiplam to the identified site.

Conclusions:

• Tether-seq is a powerful tool for discovering RNA-small molecule interactions throughout the transcriptome.
• The method provides insights into the thermodynamic properties governing small molecule binding to RNA.
• This approach can accelerate the development of RNA-targeting therapeutics.