RNA-catalyzed evolution of catalytic RNA

Area of Science:

• Origin of Life Studies
• Molecular Evolution
• Biochemistry

Background:

• Directed evolution has yielded RNA polymerase ribozymes capable of RNA replication.
• Previous ribozyme versions lacked sufficient fidelity for sustained information propagation.
• Hammerhead ribozymes are model systems for studying RNA-based evolution.

Purpose of the Study:

• To assess the impact of RNA polymerase fidelity on Darwinian evolution of RNA.
• To investigate the evolution of hammerhead ribozymes using high-fidelity replication.
• To understand the role of replication fidelity in maintaining heritable information.

Main Methods:

• Directed evolution of an RNA polymerase ribozyme for improved fidelity.
• Parallel evolution experiments using low-fidelity and high-fidelity polymerases.
• Deep sequencing to track sequence changes and fitness over evolutionary time.
• Selection for RNA cleavage activity to drive evolution.

Main Results:

• The high-fidelity polymerase successfully replicated hammerhead ribozymes, leading to evolved variants with increased fitness.
• Low-fidelity replication resulted in loss of hammerhead functionality and sequence divergence.
• Evolved hammerhead variants exhibited improved replicability, balancing a slight decrease in catalytic activity.
• Deep sequencing revealed a progressive divergence of sequences correlating with increased fitness.

Conclusions:

• High replication fidelity is essential for maintaining and evolving functional RNA in a Darwinian system.
• Achieving high fidelity is a critical bottleneck for recreating RNA-based life in the laboratory.
• This work provides insights into the potential evolutionary pathways of early RNA-based life.