What question did this study set out to answer?

The research aims to engineer aminoacyl-tRNA synthetases that can incorporate noncanonical monomers into proteins efficiently.

March 1, 2026

Co-Translational Incorporation of ( R )- and ( S )-β 2 -Hydroxyacids In Vivo : Directed Evolution of Efficient Aminoacyl-tRNA Synthetases

Key Points

The research aims to engineer aminoacyl-tRNA synthetases that can incorporate noncanonical monomers into proteins efficiently.
Developed START, a translation-independent selection platform for aaRS mutants
Applied START to engineer MaPylRS mutants to acylate tRNA with non-α-amino acid substrates
Used barcoded tRNAs to track distinct aaRS mutants in a library
Assessed efficiency and selectivity of engineered aaRS mutants across various substrates
Achieved successful incorporation of both (R)- and (S)-β2-hydroxy acids into proteins
Demonstrated that engineered aaRS mutants can accommodate diverse noncanonical substrates
Findings indicate that eukaryotic translation machinery can incorporate non-α-backbones efficiently
Several mutants displayed strong polyspecificity without recognizing α-amino acids

Abstract

Expanding the genetic code of living cells with noncanonical monomers (ncMs) relies on engineered aminoacyl-tRNA synthetases (aaRS) and their cognate tRNAs. Conventional aaRS engineering strategies rely on translation-dependent selection systems, limiting their utility for ncMs that are poorly accommodated by the native translational machinery. To address this limitation, we recently developed START, a translation-independent platform that selects Methanomethylophilus alvus pyrrolysyl-synthetase (MaPylRS) mutants based on their ability to acylate cognate tRNAMaPyl. START uses barcoded tRNAs to encode the identity of distinct aaRS mutants in a library. Acylation by active aaRS mutants protects the corresponding tRNAs from periodate oxidation, and their identity is retrieved subsequently through sequencing. START was previously applied to genetically encode noncanonical α-amino acids. Here, we successfully applied START to engineer MaPylRS mutants capable of acylating tRNAMaPyl with diverse non-α-amino acid substrates with good efficiency and fidelity, including (R) and (S) enantiomers of a β2-hydroxy acid, a β2-amino acids, and a malonate. Several mutants exhibit notable polyspecificity across noncanonical backbones while maintaining selectivity against their α-amino acid counterparts. Using these novel enzymes, we demonstrate the ribosomal incorporation of both (R)- and (S)-β2-hydroxy acids into a luciferase reporter protein expressed in Escherichia coli with good efficiency and fidelity. These results imply that highly active engineered aaRS/tRNA pairs can overcome the recently established limitations of EF-Tu with respect to non-α-amino acid substrates. The engineered MaPylRS mutants also enabled the successful incorporation of both (R)- and (S)-β2-hydroxy acids into a protein expressed in mammalian cells, demonstrating for the first time that eukaryotic translation can accommodate non-α-backbones.

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Co-Translational Incorporation of ( R )- and ( S )-β 2 -Hydroxyacids In Vivo : Directed Evolution of Efficient Aminoacyl-tRNA Synthetases

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Abstract

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