Ribosomal synthesis of nonnatural biopolymers in microscales

Summary form only given. The genetic code is established by the aminoacylation reactions by which the individual amino acids are attached to the 3´-terminal hydroxy groups of cognate transfer RNAs (tRNAs) via ester bonds. Aminoacyl-tRNA synthetases (ARSs), the enzymes responsible for catalyzing aminoacylation, are highly specific to each cognate pair of amino acids and tRNAs. Therefore, polypeptides synthesized by the translation apparatus generally contain only naturally occurring amino acids. However, when appropriate mischarged tRNAs with nonnatural amino acids are used to reassign the codons, these nonnatural ones can also be incorporated to the polypeptide chain. We here show a new concept, reprogramming the genetic code, where the genetic triplets assigned to natural amino acids are reassigned to exogenous nonnatural ones, enables mRNA-directed synthesis of nonnatural polypeptides. To achieve this, we developed a new artificial tRNA acylation system based on RNA catalysts (ribozymes), called flexizyme system. The flexizymes have ability to catalyze tRNA acylation with nearly unlimited kinds of amino acids and hydroxy acids´. By means of this flexizyme system, we have demonstrated site-directed incorporation of the amino/hydroxy acids into a model protein, green fluorescent protein, through amber/programmed frame-shift suppression using cell-free translation systems. Moreover, the flexizyme technology enables to reassign the genetic code of natural amino acids to nonnatural ones. Thus, its coupling with reconstituted cell-free translation system (called PURE system) has given de novo mRNA-encoded synthesis of nonnatural polypeptides. Furthermore, utilizing the concept of the genetic code reprogramming, we demonstrated ribosomal synthesis of nonnatural polyesters encoded in mRNA templates. Our demonstrations would open new avenues for the ribosomal synthesis of nonnatural biopolymers