Extensive breaking of genetic code degeneracy with non-canonical amino acids.

Genetic code expansion (GCE) offers many exciting opportunities for the creation of synthetic organisms and for drug discovery methods that utilize in vitro translation. One type of GCE, sense codon reassignment (SCR), focuses on breaking the degeneracy of the 61 sense codons which encode for only 20 amino acids. SCR has great potential for genetic code expansion, but extensive SCR is limited by the post-transcriptional modifications on tRNAs and wobble reading of these tRNAs by the ribosome. To better understand codon-tRNA pairing, here we develop an assay to evaluate the ability of aminoacyl-tRNAs to compete with each other for a given codon. We then show that hyperaccurate ribosome mutants demonstrate reduced wobble reading, and when paired with unmodified tRNAs lead to extensive and predictable SCR. Together, we encode seven distinct amino acids across nine codons spanning just two codon boxes, thereby demonstrating that the genetic code hosts far more re-assignable space than previously expected, opening the door to extensive genetic code engineering.

Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming.

The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.