Automated Orthogonal tRNA Generation Removes a Key Bottleneck in Scaling Non-Canonical Amino Acid Incorporation

Published in Nature Chemical Biology in 2024, this paper introduces an automated approach to generating orthogonal tRNAs, removing a significant bottleneck in scaling genetic code expansion to new non-canonical amino acids.

Orthogonal tRNAs are essential components in the system that incorporates ncAAs into proteins. Each new ncAA requires a matched tRNA/synthetase pair where the tRNA reads a specific reassigned codon and the synthetase charges it with the correct ncAA. Creating these tRNAs has historically been a manual, iterative process: designing candidate sequences, testing them for orthogonality against the host cell's existing tRNAs, verifying that they do not cross-react with native synthetases, and confirming that they function efficiently in translation. Each cycle takes weeks of bench work.

This paper automates that process. The team developed a computational and experimental pipeline that generates orthogonal tRNA variants systematically rather than one at a time. The automated approach identifies tRNA sequences that meet the orthogonality requirements, reducing the time from initial design to validated tRNA from weeks to days.

The impact on platform capability is multiplicative. Constructive Bio's ncAA library currently includes hundreds of non-canonical amino acids, but each new addition to the library depends on having a functional orthogonal tRNA/synthetase pair. Automating tRNA generation means the rate at which new ncAAs can be made available for therapeutic programmes is not gated by manual tRNA engineering.

This is particularly relevant for the discovery phase of drug development, where medicinal chemistry teams may want to screen multiple ncAA variants at specific positions in a peptide candidate. Faster tRNA generation translates directly into faster access to new chemistries, shorter timelines from target identification to lead optimisation, and a broader palette of molecular modifications available through BioForge fermentation.