Development of programmable RNA imaging with RNA-guided GFP via click chemistry

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• CRISPR-Cas systems provide precise nucleic acid targeting via guide RNAs.
• Developing novel tools for direct nucleic acid visualization in living cells remains a key challenge.

Purpose of the Study:

• To develop a simple, programmable RNA-guided platform for nucleic acid targeting and imaging.
• To establish an efficient method for conjugating guide nucleic acids with proteins.

Main Methods:

• Developed RNA-guided green fluorescent protein (RGG) using click chemistry.
• Conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids with azide-exposed proteins.
• Optimized RNA length and modification for effective RGG construction.

Main Results:

• Identified 30-nt RNA with 3'-DBCO modification as optimal for RGG.
• Successfully visualized nuclear-localized RNAs (NEAT1, Satellite III RNA) in living cells using RGG.
• Demonstrated RGG's programmability and efficiency for RNA imaging.

Conclusions:

• RGG is a customizable and efficient system for RNA imaging and molecular analysis.
• Direct conjugation of guide nucleic acids and proteins enables precise nucleic acid recognition.
• This approach holds potential for dynamic molecular modification in living cells.