Alexandra Kent

Co-Translational Incorporation of (R)- and (S)-β2-Hydroxyacids In Vivo: Directed Evolution of Efficient Aminoacyl-tRNA Synthetases 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.

Chintan's work demonstrating the efficient incorporation of non-α-amino acid backbones into proteins expressed in both E. coli and mammalian cells just came out! A great collaborative effort from @cgemcci.bsky.social! pubs.acs.org/doi/10.1021/...

Co-Translational Incorporation of (R)- and (S)-β2-Hydroxy Acids In Vitro: A Structural and Biochemical Study on the E. coli Ribosome Engineering the translation apparatus to accept backbone-modified amino acid analogues would enable the programmed synthesis of sequence-defined biopolymers with tunable properties. β-Hydroxy acids ar...

Check out our new paper using mass spectrometry and cryo-EM to show ribosomal incorporation and accommodation of β-hydroxy acid stereoisomers, bringing us closer to ribosomal synthesis of non-natural polymers. @c-majumdar.bsky.social @jhdcate.bsky.social
pubs.acs.org/doi/10.1021/...

Further Confirmation of the Structure of 3′-(2-Pyridyldithio)-3′-deoxyadenosine and 3′-Thio-3′-deoxyadenosine: Synthetic Convergence with Cordycepin 3′-Deoxynucleosides have demonstrated profound biochemical utility, including in our recent work on the use of aminoacyl thio-tRNAs for in vitro translation. Though our preparation of 3′-(2-pyridyldithio)-3′-deoxyadenosine─a key synthetic precursor en route to aminoacyl thio-tRNAs─followed robust prior precedents, an unrelated recent structural revision prompted us to validate the regioselectivity of our route further. Here we provide confirmatory evidence for the structure of 3′-(2-pyridyldithio)-3′-deoxyadenosine and downstream analogues via synthetic convergence with a 3′-deoxynucleoside antibiotic, cordycepin, and concomitant MicroED analysis.

✨ New from C-GEM in JOC @pubs.acs.org:

Further Confirmation of the Structure of 3′-(2-Pyridyldithio)-3′-deoxyadenosine and 3′-Thio-3′-deoxyadenosine: Synthetic Convergence with Cordycepin

Congrats to Taylor and coauthors! #NSFfunded
pubs.acs.org/doi/10.1021/...

Escherichia coli ribosomes support translation of (R) and (S) β2-hydroxyacids in vitro: a structural and biochemical study The ribosomal incorporation of backbone-modified amino acid analogs into peptides and proteins enables the programmed synthesis of sequence-defined biopolymers with tunable properties. However, the su...

Check out our latest preprint describing two structures of ribosomes complexed with the two enantiomers of a β2-hydroxy acid. Our structures show that despite stereochemical differences, both are ultimately well positioned for bond formation within the ribosome!
chemrxiv.org/engage/chemr...

📰 Great "First Reactions" by @aprillukowski.bsky.social featuring C-GEM's recent thioribose work!

pubs.acs.org/doi/10.1021/...