SHAPE-Based Chemical Probes for Studying preQ₁-RNA Interactions in Living Bacteria

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Interrogating RNA-small molecule interactions within cells is crucial for RNA-targeted drug discovery.
• Identifying small molecule-bound RNAs and their binding sites in vivo is essential for assessing on-target activity in drug development.
• Existing chemical probing technologies require enhancement for cellular applications.

Purpose of the Study:

• To develop a chemical probing strategy for interrogating RNA-small molecule interactions inside living cells.
• To establish a method for identifying small molecule-bound RNAs and mapping their binding sites.
• To support the development of RNA-targeted therapeutics.

Main Methods:

• Development of an icSHAPE-based probe for a preQ1 riboswitch.
• Utilizing RNA acylation chemistry for cellular probing.
• Employing mutational profiling (MaP) to map binding sites on RNA.

Main Results:

• The developed icSHAPE-based preQ1 probe demonstrated biological activity in a riboswitch reporter assay.
• The probe successfully enriched the preQ1 riboswitch from living bacterial cells.
• The preQ1 binding site on the riboswitch RNA was mapped using MaP.

Conclusions:

• The described chemical probing strategy effectively interrogates cellular RNA-small molecule interactions.
• This method enables the identification of small molecule-bound RNAs and their binding sites in vivo.
• The strategy supports the advancement of RNA-targeted drug discovery and the development of novel therapeutics.