A versatile cis-blocking and trans-activation strategy for ribozyme characterization

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Ribozyme-based synthetic RNA control devices regulate cellular functions via environmental signals.
• Accurate measurement of ribozyme cleavage rates is crucial for optimizing these gene-regulatory devices.
• Generating full-length RNA for characterization is challenging due to in vitro cleavage during transcription.

Purpose of the Study:

• To develop an efficient, gel-free method for scalable generation of functional full-length ribozyme-encoding RNA.
• To establish a rapid, label-free assay for real-time monitoring of ribozyme cleavage.
• To advance the characterization and engineering of ribozyme-based gene-regulatory devices.

Main Methods:

• A two-step gel-free process involving cis-blocking and trans-activation was developed for RNA generation.
• The strategy was validated using natural and synthetic ribozymes.
• Surface plasmon resonance (SPR) was employed to create a label-free, real-time cleavage assay.

Main Results:

• The developed method enables scalable production of full-length ribozyme-encoding RNA without laborious gel separation.
• Cleavage rate constants obtained using the new method are comparable to traditional approaches.
• The SPR assay provides continuous, real-time monitoring of ribozyme activity.

Conclusions:

• The novel cis-blocking and trans-activation strategy offers a simplified and scalable approach to generating functional full-length ribozyme RNA.
• The SPR-based assay complements this strategy, enhancing the characterization of ribozyme devices.
• These advancements will accelerate the design and optimization of synthetic RNA control systems.