RNA-binding fluorogenic probes: G-clamp conjugated with a thiazole orange derivative for screening RNA-binding small...

Area of Science:

• Molecular Biology
• Drug Discovery
• Chemical Biology

Background:

• RNA is a key target for drug discovery, necessitating effective methods for identifying RNA-binding small molecules.
• Fluorescent indicator displacement (FID) assays are crucial for screening these molecules, but require diverse indicators to avoid missing potential hits.

Purpose of the Study:

• To develop novel RNA-binding fluorogenic molecular probes for enhanced FID assays.
• To create probes with distinct RNA-binding modes compared to existing indicators to broaden screening capabilities.

Main Methods:

• Conjugation of thiazole orange (TO) derivatives to the G-clamp RNA-binding molecule to create TO-G-clamp probes.
• Synthesis and evaluation of four TO-G-clamp analogs.
• Utilizing FID assays and SHAPE-MaP analysis to assess probe performance and RNA-binding sites.

Main Results:

• Four TO-G-clamp analogs were synthesized, all maintaining the broad RNA-binding selectivity of G-clamp.
• TO-G-clamp-Bn demonstrated high consistency with G-clamp's RNA-binding selectivity and identified unique hit compounds missed by TO-PRO-1.
• SHAPE-MaP analysis indicated that TO-G-clamp binds near the binding sites of identified hit compounds like AZ191.

Conclusions:

• The novel TO-G-clamp probes, particularly TO-G-clamp-Bn, offer a valuable addition to FID assays for RNA-targeted drug discovery.
• These probes enable the identification of unique small molecules with distinct RNA-binding properties.
• The findings advance the development of more comprehensive screening strategies for RNA-binding drug candidates.