Engineering covalent small molecule-RNA complexes in living cells.

Area of Science:

• Molecular Biology
• Biochemistry
• Chemical Biology

Background:

• Covalent labeling of RNA in living cells is challenging.
• Existing methods lack precision and versatility for studying RNA dynamics.

Purpose of the Study:

• To develop a structure-guided approach for engineering covalent RNA aptamer-ligand complexes.
• To enable precise labeling and monitoring of RNA in vitro and in vivo.
• To demonstrate the utility of this strategy for live-cell imaging and drug targeting.

Main Methods:

• Modifying RNA ligands with electrophilic handles for covalent attachment to specific RNA guanines.
• Utilizing the preQ1-I riboswitch as a model system for validation.
• Developing a covalent fluorescent light-up aptamer (coPepper) for imaging applications.
• Employing fluorescence recovery after photobleaching (FRAP) to study intracellular RNA dynamics.
• Introducing bioorthogonal handles for RNA pull-down experiments.

Main Results:

• Successfully engineered covalent RNA aptamer-ligand complexes.
• Demonstrated in vitro and in vivo labeling of the preQ1-I riboswitch.
• The coPepper system exhibits stable fluorescence in live-cell imaging and super-resolution microscopy.
• coPepper is suitable for FRAP to monitor RNA dynamics.
• A dual-handle ligand facilitates traceable pull-down of covalently linked RNA.
• The tethering strategy shows promise for drug targeting applications.

Conclusions:

• The structure-guided approach provides a versatile platform for covalent RNA labeling and manipulation.
• Engineered covalent complexes enable advanced RNA imaging and dynamic studies.
• This strategy holds potential for therapeutic interventions and molecular diagnostics.