Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons

Abstract

The genetic code in most organisms codes for 20 proteinogenic amino acids or translation stop. In order to encode more than 20 amino acids in the coding system, one of stop codons is usually reprogrammed to encode a non-proteinogenic amino acid. Although this approach works, usually only one amino acid is added to the amino acid repertoire. In this study, we incorporated non-proteinogenic amino acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA(Arg) in a cell-free protein synthesis system by using a tRNA(Arg) -specific tRNase, colicin D. Then by supplementing the system with tRNACCU , the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 proteinogenic amino acids.

Rational Design of Aptamer-Tagged tRNAs

“…Alternatively, some of the endogenous tRNAs or tRNA-aaRS pairs might be replaceable with allo-tRNAs or orthogonal pairs composed of allo-tRNAs [ 60 , 63 , 64 , 65 ]. For reprogramming the genetic code, the allo-tRNA sequestration system may be useful for use in vivo and in vitro [ 66 ] and for the purification and elimination of allo-tRNA molecules from tRNA mixtures [ 66 , 67 , 68 , 69 ]. Application of the allo-tRNA chassis may not be limited to E. coli but may possibly need further optimization, especially for their use in archaea and eukaryotes due to the difference in the manner of tRNA Ser recognition by SerRS [ 70 , 71 ].…”

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…Colicin D specifically cleaves tRNA Arg at the anticodon loop, including tRNA , tRNA , tRNA , tRNA , tRNA , and tRNA , which can free six codons for reassignment ( Tomita et al, 2000 ). Kang et al utilized resin-bound colicin D to cleave all tRNA Arg in the cell-free system and supplemented synthetic tRNA for arginine translation at a single codon, successfully demonstrating the recoding of four sense codons to ncAAs ( Lee K.B. et al, 2016 ).…”

Exploring sequence space: harnessing chemical and biological diversity towards new peptide leads

“…[25] Using an orthogonal approach, instead of artificially transcribing all tRNAs, Kang and coworkers selectively depleted all E. coli tRNA Arg with colicin D to divide the arginine codon box. [26] Integrating the FIT system with mRNA display gave rise to the random non-standard peptide integrated discovery (RaPID) system. [27] The versatility of this system is reflected in its broad use in the pharmaceutical industry today.…”