Bioorthogonal Cyclopropenones for Investigating RNA Structure

Area of Science:

• Molecular Biology
• Chemical Biology
• Biochemistry

Background:

• RNA structure is crucial for protein production and cellular functions.
• Misfolded RNAs are implicated in various diseases, but their structures are not fully understood.
• Current methods for studying RNA structures in native environments have limitations, including high background noise.

Purpose of the Study:

• To develop a novel, chemically triggered method for interrogating RNA structures in native cellular environments.
• To establish more accurate RNA structure-function relationships.
• To overcome the limitations of existing RNA probing tools.

Main Methods:

• Development of bioorthogonal cyclopropenones (CpOs) as chemical triggers for RNA cross-linking.
• Conjugation of a CpO motif to thiazole orange (TO-1) to create TO-1-CpO probe.
• Application of TO-1-CpO to a model RNA aptamer (Mango) and subsequent chemical triggering with phosphines to induce cross-linking.

Main Results:

• TO-1-CpO demonstrated selective binding to the Mango RNA aptamer with nanomolar affinity, indicated by fluorescence.
• Chemical triggering with phosphines successfully induced covalent cross-linking between the CpO and RNA.
• Cross-linking efficiency was shown to be dependent on both time and reagent dose.

Conclusions:

• The study presents a novel, chemically triggered approach for RNA cross-linking using bioorthogonal cyclopropenones.
• This method provides a valuable new tool for studying RNA structures and conformations in native biological settings.
• The developed probes expand the available toolkit for RNA research, potentially advancing our understanding of RNA function and disease.