A Small-Molecule-Based Approach to Sense Codon-Templated Natural-Unnatural Hybrid Peptides. Selective Silencing and Reassignment of the Sense Codon by Orthogonal Reacylation Stalling at the Single-Codon Level

Abstract

In the presence of the stable sulfamoyl analogue of phenylalanyl adenylate (Phe-SA), the UUU/UUC sense codon for phenylalanine (Phe) can be silenced and reassigned to a naphthylalanine (Nap) conjugated to tRNAPhe. We have demonstrated the efficiency and selectivity or orthogonality of the Phe-to-Nap reassignment induced by an "orthogonal reacylation stalling" strategy at the single-codon level in the translation of mRNAs of dihydrofolate reductase and a 24-mer oligopeptide. We used a prokaryotic translation system with an essential preincubation, during which the endogenous precharged phenylalanyl-tRNAPhe undergoes deacylation and the reacylation of the resulting tRNAPhe is stalled by the action of Phe-SA to inhibit the phenylalanyl-tRNA synthetase activity. We discuss the significance of the present small-molecule-based approach to sense-codon templated natural-unnatural peptides.

Non‐Canonical Amino Acids in Protein Polymer Design

“…8 The fidelity of the method is enhanced by inhibition of the phenylalanyltRNA synthetase. 8,9 In order to reduce competition with the natural amino acids, and to effect site-specific insertion of non-natural amino acids, several research groups in the 1980s turned to suppressor tRNAs. 10,11 The genetic code includes three "stop" or "nonsense" codons that signal termination of the growing polypeptide chain-the amber, opal, and ochre codons.…”

Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Acids into Polypeptides

“…The stop codons [ 12 ] and quadruplet codons [ 8 , 13 ], which are often used as suppressor codons in genetic code expansion methods, generally suffer from undesired termination and/or incorporation of canonical amino acids, because RFs and endogenous cognate or near-cognate AA-tRNAs competitively read these codons [ 14 ]. In the case of in vitro engineering of translation, simple depletion of competing factors, such as canonical amino acids, AARSs, and RFs from the reaction mixture, has succeeded to suppress the undesired background reactions [ 10 , 15 , 16 , 17 , 18 ]. On the other hand, suppression of the competitions in in vivo genetic code expansion has been more demanding.…”

Genetically Encoded Libraries of Nonstandard Peptides

“…Other approaches to decrease competitive translation events against nonproteinogenic amino acid incorporation in vitro include heat inactivation of RF-1 [84], aptamer-mediated RF-1 inhibition [85], and antibody-mediated RF-1 inhibition [86], yet none of these resulted in complete codon reassignment. Another interesting approach is the pretreatment of a lysate translation system with a phenylalanyl-tRNA synthetase (PheRS) inhibitor, and nearly complete reassignment of the Phe sense codon to naphthylalanine was demonstrated, although its general application to multiple codons has yet to be reported [87]. …”