Engineered triply orthogonal pyrrolysyl–tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids

Incorporating one non-canonical amino acid into a protein is well established. Incorporating two or three different ncAAs into the same molecule, each at a defined position, required new molecular machinery. This 2020 Nature Chemistry paper from the Chin group at the MRC Laboratory of Molecular Biology delivered that advance by engineering three mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs.

Orthogonality is the critical requirement. Each engineered synthetase/tRNA pair must recognise only its assigned non-canonical amino acid and only its assigned codon. It must not cross-react with canonical amino acids, other ncAAs in the system, or other tRNAs. When multiple pairs operate simultaneously in the same cell, cross-reactivity would produce misincorporation: the wrong amino acid at the wrong position, destroying the precision that makes site-specific ncAA incorporation therapeutically useful.

The team engineered three pyrrolysyl-tRNA synthetase (PylRS) variants, each derived from a different archaeal species, that function independently of one another and independently of the host cell's existing translation machinery. Each pair was validated for mutual orthogonality: synthetase A charges only tRNA A with ncAA A, synthetase B charges only tRNA B with ncAA B, and synthetase C charges only tRNA C with ncAA C, with no measurable cross-talk between them.

This work is what makes multi-ncAA therapeutics possible. Constructive Bio's platform can incorporate up to three different non-canonical amino acids into a single peptide or protein: for example, a click-chemistry conjugation handle, a protease-resistant backbone modification, and a lipidation site for half-life extension, all in one molecule. Each ncAA is placed at a genetically specified position with the precision of a DNA sequence change.

The triply orthogonal system underpins the molecular diversity that distinguishes BioForge-manufactured therapeutics from what is achievable by solid-phase peptide synthesis, where incorporating even a single ncAA at scale presents significant challenges.