Ribozyme-Catalyzed Late-Stage Functionalization and Fluorogenic Labeling of RNA

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Site-specific RNA modification is crucial for attaching labels like fluorophores.
• Aldehydes are versatile functional groups for bioconjugation reactions.

Purpose of the Study:

• To develop a ribozyme-based method for synthesizing aldehyde-functionalized RNA.
• To demonstrate the utility of this method for RNA labeling and functionalization.

Main Methods:

• Utilized the methyltransferase ribozyme MTR1 as an alkyltransferase.
• Site-specific N1 benzylation of purine followed by ring opening and hydrolysis.
• Conjugated aldehyde-reactive probes (biotin, fluorescent dyes) to modified RNAs.

Main Results:

• Successfully synthesized aldehyde-functionalized RNA with a 5-amino-4-formylimidazole residue.
• Demonstrated efficient conjugation of biotin and fluorescent dyes to short RNAs and tRNA.
• Generated a novel hemicyanine chromophore directly on RNA via fluorogenic condensation.

Conclusions:

• The MTR1 ribozyme can be repurposed for site-specific RNA functionalization.
• This method provides a new tool for late-stage RNA modification and labeling.
• Expands the application of ribozymes in bioorthogonal chemistry.