Bioorthogonal cyclopropenones for investigating RNA structure

Area of Science:

• Molecular Biology
• Chemical Biology
• Biochemistry

Background:

• RNA secondary and tertiary structures are crucial for cellular processes.
• Misfolded RNAs are implicated in diseases, but their mechanisms are not fully understood.
• Current methods for studying RNA structure in situ have limitations, including high background reactivity.

Purpose of the Study:

• To develop novel chemical tools for interrogating RNA structure and trapping native conformations.
• To establish more comprehensive and accurate RNA structure-function relationships.
• To overcome limitations of existing RNA crosslinking methods.

Main Methods:

• Development of chemically triggered RNA crosslinking probes using bioorthogonal cyclopropenones (CpOs).
• Synthesis of CpOs conjugated to thiazole orange (TO-1) for selective RNA binding.
• Application of phosphine-triggered crosslinking in model RNA systems.

Main Results:

• CpO-TO-1 conjugates demonstrated selective binding to a model RNA aptamer with nanomolar affinity.
• Chemically triggered crosslinking was achieved upon phosphine administration.
• Crosslinking efficiency was dependent on time and dose, and applicable to model RNAs under biologically relevant conditions.

Conclusions:

• The developed cyclopropenone-based probes offer a selective and chemically triggered approach for RNA crosslinking.
• This method expands the available toolkit for studying RNA structure and function in cellular environments.
• The findings facilitate a deeper understanding of RNA's role in biological processes and disease.