Flexizymes that enable to reprogram the genetic code

Abstract

The lecture describes a new means to reprogram the genetic code, which enables us to express “non‐standard” peptides containing multiple non‐proteinogenic amino acids. To execute the genetic code reprogramming, we developed an artificial RNA enzyme (ribozyme), referred to as “flexizyme” capable of aminoacylating tRNAs. The most notable feature of flexizyme is its versatility; it is able to charge virtually any amino acids onto tRNAs bearing various anticodons. Moreover, the flexizyme also charges amino acids with non‐proteinogenic sidechain, D‐amino acids, N‐alkyl‐amino acids, and hydroxy acids onto tRNA. Thus, the reassignment of such non‐proteinogenic amino acids to We also integrated this unique enzyme system with a specially reconstituted E. coli cell‐free transcription‐translation coupled system, from which certain amino acids (occasionally their cognate aminoacyl‐tRNA synthetases) were withdrawn to vacant the corresponding codons. By the integration of these two systems, any desired amino and hydroxy acids can be reassigned to the vacant codons, and we have recently showed that a wide variety of non‐standard peptides can be expressed from mRNAs under the reprogrammed genetic code. The lecture also describes applications of the methodology to the ribosomal generation of cyclic peptides closed by a linkage between the N‐terminus and Cys sidechain or a linkage between the N‐terminus and C‐terminus (backbone‐cyclic peptides).