Nonstandard Peptide Expression under the Genetic Code Consisting of Reprogrammed Dual Sense Codons

Abstract

We here demonstrate a translation system that is governed by a reprogrammed genetic code consisting of "dual sense codons." A dual sense codon assigns two distinct amino acids for initiation and elongation. Because multiple dual sense codons independently function without cross-readings, this system enables the expansion of the repertoire of initiators as well as elongators that can be used simultaneously.

Recoding the Genetic Code with Selenocysteine

“…Progress in the last decade allowed site-specific incorporation of one or two ncAAs into recombinant proteins by reassignment of nonsense codons. [4b] In vitro protein synthesis methods were recently enhanced by creating dual-meaning initiation and sense codons to synthesize potential chemotherapeutic peptides with two ncAAs, [20] and amino acid starvation methods permitted production of proteins with three ncAAs for biological imaging applications. [21] Furthermore, unlike the Sec machinery that only recodes codons associated with SECIS, current genetic code expansion methods are not sitespecific because they lead to global reassignment of stop codons; this contaminates the natural proteome with ncAAs, resulting in growth defects.…”

Synthesis of fused tricyclic peptides using a reprogrammed translation system and chemical modification

“…The incorporation of an L-lactic acid ( HO A) at a designated position can be achieved by genetic code reprogramming. Since we utilized the Met-deficient FIT system for the reassignment of ClAc-D W, the elongator AUG codon is left vacant; therefore, this vacant codon can be utilized for the assignment for HO A, similar to the strategy reported elsewhere [18,24]. We thus designed a DNA template (D1e) based on the sequence of D1 to include an elongator AUG codon between Cys2 and Cys3, which would be suppressed with HO A by the use of HO A-tRNA enAsn CAU .…”

Hijacking Translation Initiation for Synthetic Biology

“…For example, 17 – 26 are N α ‐modified amino acids, several of which contain reactive moieties that have been used to generate side‐chain‐to‐backbone and backbone‐to‐backbone cyclic peptides . d ‐Amino acids, which are frequently components of natural cyclic peptides, have also been used to initiate translation via flexizyme‐catalyzed acylation of tRNA fMet . One such example is 27 , whose terpene side chain containing a reactive chloroacetamide was used to generate a macrocyclic peptide inspired by the structure of the antifungal natural product amphotericin B .…”