Discovery of Selective RNA-Binding Small Molecules by Affinity-Selection Mass Spectrometry

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Noncoding RNAs (ncRNAs) are increasingly recognized for their roles in disease.
• Targeting ncRNAs with small molecules offers a promising therapeutic strategy.
• The flavin mononucleotide (FMN) riboswitch is a key ncRNA target, with ribocil as a known ligand.

Purpose of the Study:

• To apply and validate the Automated Ligand Detection System (ALIS), an indirect affinity-selection mass spectrometry (AS-MS) technique, for high-throughput screening (HTS) of small molecule-ncRNA interactions.
• To identify novel small molecules targeting the FMN riboswitch.
• To characterize the binding modes and conformational effects of identified compounds on the FMN riboswitch.

Main Methods:

• Utilized the ALIS platform, an indirect AS-MS technique, for selective detection of small molecule-ncRNA interactions.
• Performed HTS against large, unbiased small-molecule libraries to identify novel FMN riboswitch binders.
• Employed crystal structures to analyze the conformational changes induced by different small molecules in the FMN riboswitch.

Main Results:

• Successfully applied ALIS for selective detection and HTS of small molecule-ncRNA interactions.
• Identified novel compounds targeting the FMN riboswitch, distinct from FMN and ribocil.
• Observed that different small molecules induce diverse conformations of the FMN riboswitch, correlating with varied activity profiles.

Conclusions:

• The ALIS platform is validated as an effective tool for HTS of RNA-binding small molecules.
• Noncoding RNAs can be targeted by chemically diverse small molecules.
• These findings support the development of ncRNA-targeting small molecules as a broad therapeutic approach.