Site-Selective Ligand Selection by Mutational Profiling for Covalent RNA Targeting.

Area of Science:

• Chemical Biology
• Molecular Biology
• RNA Therapeutics

Background:

• RNA-targeted covalent probes are crucial for studying RNA structure and function.
• Designing selective probes for specific RNA targets remains a significant challenge.
• The FMN riboswitch is a key regulatory element in bacteria.

Purpose of the Study:

• To develop a structure-informed, bottom-up approach for designing RNA-targeted covalent probes.
• To create a highly base-selective covalent probe targeting the FMN riboswitch.
• To validate the probe's selectivity and application in complex RNA environments.

Main Methods:

• Utilized mutational profiling and a structure-informed design strategy.
• Synthesized a library of covalent probes based on the Ribocil scaffold.
• Screened probes against FMN riboswitch aptamers from diverse bacterial species.
• Validated probe selectivity and covalency using competitive photo-affinity labeling and mass spectrometry.

Main Results:

• Identified Covacil, a highly base-selective covalent probe for the FMN riboswitch aptamer.
• Covacil achieves covalent modification at low micromolar concentrations within 10 minutes.
• Demonstrated >1,000-fold increased base selectivity compared to non-targeted probes.
• Confirmed Covacil's selective reactivity towards the FMN riboswitch in total RNA samples.

Conclusions:

• The developed structure-informed approach enables the design of potent and selective RNA-targeted covalent probes.
• Covacil represents a valuable tool for studying FMN riboswitch function and RNA biology.
• Covacil's base-selective reactivity in total RNA highlights its potential for transcriptomic analysis.