Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genetics

Background:

• Genetic code expansion technologies aim to increase the diversity of amino acids incorporated into proteins in vivo.
• Current methods often rely on non-native translational machinery and suffer from low efficiency.

Purpose of the Study:

• To engineer Escherichia coli transfer RNAs (tRNAs) capable of translating quadruplet codons.
• To enhance the efficiency and reliability of quadruplet codon translation in a bacterial system.

Main Methods:

• Developed a library-cross-library selection to identify functional quadruplet codon-anticodon pairs.
• Employed phage-assisted continuous evolution (PACE) to evolve quadruplet-decoding tRNAs (qtRNAs).
• Assessed the stability, aminoacylation, and translational processivity of evolved qtRNAs.

Main Results:

• Identified novel quadruplet codon-anticodon pairs.
• Achieved up to an 80-fold increase in quadruplet codon translation efficiency using the qtRNA-PACE strategy.
• Demonstrated multiplexed decoding of up to four unique quadruplet codons in a single reporter system.
• Evolved qtRNAs maintained base pairing, were aminoacylated by cognate synthetases, and supported processive translation.

Conclusions:

• Escherichia coli tRNAs can be engineered and evolved to decode quadruplet codons.
• The developed qtRNA system enables efficient and multiplexed genetic code expansion.
• This work lays the foundation for a future translation system based entirely on quadruplet codons.