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

摘要

The ribosome is a highly sophisticated RNA machine that directs the mRNA-templated process of amino acid polymerization,which ultimately produces size-,shape-,and sequence-defined polymers (i.e.,proteins).The previous demonstration of the "adapter hypothesis" and intensive work to incorporate unnatural substrates with chemically misacylated tRNAs have led to the current challenge of using in vivo or in vitro sense codon-decoding translation systems to synthesize sequence-programmed peptidomimetics and unnatural biopolymers.However,because of the unavoidable competition with endogenous tRNA,effective in vitro sense codon suppression has been limited to rare or minor codons.More abundant codons could also be suppressed with misacylated tRNAs to give fully unnatural peptidomimetics but only in endogenous RS/tRNA-free or tRNA-depleted translation systems.Although these elaborate systems allow sense codon-based decoding,the reconstitution and modification procedures require specialized techniques,thus severely restricting their broad applications.We designed a different and simple route,an in situ "orthogonal reacylation stalling" method,to achieve sense codon reassignment.This new route is based on the successful demonstration that the translation pauses codon-selectively in a cell-free system prepared from specific tRNA synthetase (RS)-defective E.coli strains.