Growth, Dissolution and Segregation of Genetically Encoded RNA Droplets by Ribozyme Catalysis

Area of Science:

• * Synthetic Biology
• * Biophysics
• * Materials Science

Background:

• * Active droplets, membraneless compartments, are key models for early life and synthetic cells.
• * Programming dynamic behaviors with genetic information is crucial for evolvable systems.

Purpose of the Study:

• * To engineer transiently active RNA droplets with sequence-programmable dynamics.
• * To establish a genetically encoded life cycle for synthetic protocells.
• * To develop methods for observing and controlling droplet behavior over time.

Main Methods:

• * Integrating ribozyme catalysis sites into RNA nanostar sequences for self-assembly.
• * Trapping individual droplets in hydrogel cages via in situ photopolymerization for observation.
• * Utilizing different ribozymes (hammerhead, hairpin) to control degradation kinetics.
• * Employing sequence-specific RNA cleavage to trigger droplet segregation.
• * Encapsulating DNA templates for droplet regrowth.

Main Results:

• * Successfully created transiently active RNA droplets with sequence-controlled dissolution.
• * Demonstrated programmable degradation rates using hammerhead (fast) and hairpin (slow) ribozymes.
• * Achieved segregation of mixed droplet populations through sequence-specific cleavage.
• * Established a proof-of-principle for a minimal, genetically encoded cycle of droplet dissolution and regrowth.
• * Linked RNA sequence directly to droplet stability, composition, and life-cycle dynamics.

Conclusions:

• * Developed a robust platform for engineering evolvable materials.
• * Advanced the bottom-up construction of synthetic cells with programmable life cycles.
• * Demonstrated the potential for genetic information to control the behavior of active droplets.