In vitro ribosome synthesis and evolution through ribosome display

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Directed evolution of ribosomes is hindered by the need for cell viability, limiting mutation possibilities.
• Developing novel ribosome functions requires overcoming these cellular constraints.

Purpose of the Study:

• To develop a cell-free, in vitro methodology for ribosome synthesis and evolution (RISE).
• To enable directed evolution of ribosomes independent of cell viability.
• To facilitate the selection of ribosomes with expanded substrate incorporation and novel functions.

Main Methods:

• Combined cell-free synthesis and assembly of translationally competent ribosomes.
• Utilized ribosome display for a fully in vitro evolution system.
• Screened large libraries of ribosomal RNA (rRNA) variants.

Main Results:

• Successfully selected active rRNA genotypes from a library of ~1.7 × 10^7 variants.
• Identified clindamycin-resistant mutant ribosomes from a library of ~4 × 10^3 variants.
• Demonstrated the prevalence of positive epistasis in resistant genotypes, crucial for new function selection.

Conclusions:

• The Ribosome Synthesis and Evolution (RISE) method provides a powerful platform for in vitro ribosome evolution.
• RISE overcomes cell viability limitations, enabling broader exploration of ribosome function.
• This methodology will advance the understanding of molecular translation and facilitate the engineering of novel ribosome properties.