SHAPE-based chemical probes for studying preQ₁-RNA interactions in living bacteria

Area of Science:

• Chemical biology
• RNA biology
• Drug discovery

Background:

• Interrogating RNA-small molecule interactions is vital for RNA-targeted drug discovery.
• Identifying small molecule-bound RNAs and their binding sites in cells is essential for hit-to-lead campaigns.

Purpose of the Study:

• To develop a chemical probing strategy to identify and map RNA-small molecule interactions within living cells.
• To demonstrate the utility of this strategy using the preQ1 metabolite and its riboswitch RNA as a model system.

Main Methods:

• Development of an in vivo click selective 2'-hydroxyl acylation analyzed by primer extension (icSHAPE)-based probe.
• Utilizing the probe in a preQ1 riboswitch reporter assay to confirm biological activity.
• Enriching the preQ1 riboswitch from bacterial cells using the developed probe.
• Mapping the preQ1 binding site on the riboswitch RNA using mutational profiling (MaP).

Main Results:

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

Conclusions:

• This chemical probing strategy enables the interrogation of cellular RNA-small molecule interactions.
• The method supports the development of RNA-targeted therapeutics by identifying on-target activity.
• This approach fills a technological gap in studying RNA-small molecule interactions in complex cellular environments.