Rare codon recoding for selective and efficient noncanonical amino acid incorporation [RNA-seq]

The ability to genetically encode noncanonical amino acids (ncAAs) has empowered proteins with improved or novel properties. However, existing strategies in mammalian cells rely on the introduction of blank codon for incorporating ncAAs, which is very inefficient and limits their widespread applications. Here, we develop a rare codon recoding strategy that takes advantage of the relative rarity of the TCG codon to achieve highly selective and efficient ncAA incorporation through systematic engineering and big data model predictions. We highlight the broad utility of this strategy for the incorporation of dozens of ncAAs into various functional proteins at the wild-type protein expression levels, as well as the synthesis of proteins with up to 6-site ncAAs or 4 distinct ncAAs in mammalian cells for downstream applications. Overall design: Given the high recoding efficiency of the engineered TCG recoding system, we wondered whether it would lead to high background incorporation in the proteome and affect cell viability. For this purpose, We perform the RNA-seq to examine the transcriptomics of cells tranfected with TCG recoding system (RTS). Three groups were setted. Sample-1 was HEK293T cells transfected with TCG recoding system dubbed as "RTS". Sample-2 was HEK293T cells transfected with TCG recoding system and EGFP-151TCG reporter named as "RTS-Rep" . Sample-3 was HEK293T cells transfected with blank plasmid vector named as "Vec". The three samples were subjected to RNA-Seq to analyze the global difference in expression level.