Recombination, RNA evolution, and bifunctional RNA molecules isolated through chimeric SELEX

Area of Science:

• Molecular Biology
• RNA Engineering
• Origin of Life Studies

Background:

• RNA recombination can generate novel functions and may be crucial for early life evolution.
• Assessing RNA functional retention after recombination is key to understanding its benefits or detriments.

Purpose of the Study:

• To engineer dual-function RNAs by recombining structural domains.
• To investigate the impact of recombination on RNA binding activity and folding.
• To develop selection methods for optimizing bifunctional RNA molecules.

Main Methods:

• Fusing aptamers selected for specific targets (coenzyme A, chloramphenicol, adenosine).
• Applying dual selection pressure to recombined RNA populations.
• Analyzing binding activity in solution and on affinity resins.
• Conducting deletion/selection experiments to confirm domain integrity.

Main Results:

• Chimeric RNAs showed reduced binding to both targets due to potential misfolding.
• Dual selection pressure successfully identified combinations with improved dual-target binding.
• Reselected RNAs exhibited better folding and binding activity compared to arbitrarily combined aptamers.
• Binding sequences were confirmed to be contained within their respective domains, supporting modularity.

Conclusions:

• RNA recombination is a viable strategy for engineering bifunctional molecules with potential applications in catalysis and therapeutics.
• The combinatorial approach effectively generates a vast number of dual-function RNAs.
• The modular nature of RNA domains facilitates engineering of complex functionalities.