FLARE: a label-free fluorescence-assisted method for RNA engineering of three-way junctions

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Structure and Engineering

Background:

• RNA three-way junctions (3WJs) are critical structural motifs influencing RNA function.
• Thermal stability of RNA 3WJs is vital for biological roles, but engineering methods are lacking.
• Enhanced RNA stability can improve native folding and overall functionality.

Purpose of the Study:

• To develop a high-throughput method for engineering RNA three-way junctions.
• To enhance the thermal stability and functionality of RNA 3WJs.
• To demonstrate the broad applicability of the developed engineering method.

Main Methods:

• Developed a label-free, fluorescence-assisted method for RNA engineering (FLARE).
• Utilized the Baby Spinach aptamer and Phi29 bacteriophage pRNA 3WJ scaffold.
• Engineered a 5S ribosomal RNA 3WJ for enhanced thermal stability.

Main Results:

• FLARE successfully engineered an RNA 3WJ with significantly enhanced thermal stability.
• The engineered 5S 3WJ improved CRISPR-Cas9 single guide RNA (sgRNA) cleavage efficiency.
• The engineered 3WJ was utilized to create RNA nanotriangles for potential therapeutic applications.

Conclusions:

• FLARE provides a versatile platform for engineering RNA 3WJs across various applications.
• Engineered RNA 3WJs can enhance the performance of RNA-based technologies like CRISPR-Cas9.
• This method facilitates the study and engineering of functional RNAs, including novel discoveries.