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

Phillip Yesley1, Georgia Poulladofonou1, Danny Incarnato2

  • 1Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen, 6525 AJ, Netherlands.

Angewandte Chemie (International Ed. in English)
|December 24, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed Covacil, a novel RNA-targeted covalent probe. This probe selectively modifies the FMN riboswitch, enabling precise RNA analysis within complex biological samples.

Keywords:
Covalent modificationNucleic‐acid chemistryRNA probingRNA targetingRiboswitch

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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.