Prior to the emergence of the contemporary biosphere, the first replicating systems are thought to have progressed through an RNA-based stage. Such...

Ribosomes are capable of incorporating a wide array of natural and unnatural monomers into growing polymer chains, but can be stalled by monomers with constrained or non-natural backbones. Here we eva...

Researchers in the Cate and Schepartz Groups at the University of California, Berkeley, and the Lawrence Berkeley National Laboratory, published in the <em>Journal of the American Chemical Society</em...

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.

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...

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.

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...

Thioesters, rather than oxo-esters, can be tolerated and processed during translation to incorporate unnatural monomers.