Fig. 1: Overview of the X–Y expanded genetic code technology.
From: An engineered IL-2 reprogrammed for anti-tumor therapy using a semi-synthetic organism
a Structures of the natural base pairs A-T (left) and G-C (center) and the X–Y synthetic DNA base pair (right, highlighted in orange/blue). Rather than utilizing hydrogen bond donors and acceptors between pairs (dotted lines), the synthetic base pair formed between NaM (X) and TPT3 (Y) utilizes non-Watson–Crick mode of pairing that is based predominantly on hydrophobic packing interactions. b Schematic of the semi-synthetic organism (SSO) platform based on E. coli BL21(DE3). The organism constitutively expresses a variant of the PtNTT2 nucleotide transporter (green)11, which mediates the transport of X and Y nucleoside triphosphates from the growth medium into the cytoplasm for DNA and RNA synthesis. X–Y containing DNA sequences are transcribed into mRNA and tRNA that contain the codon (AXC) and anticodon sequences (GYU), respectively. The orthogonal tRNA synthetase PylRS from M. barkeri (blue) specifically recognizes the AzK (red shape) provided in the culture medium and charges this residue onto the corresponding GYU anticodon-containing orthogonal pylT tRNA. Finally, the cell’s ribosomal translation machinery utilizes the resulting pools of AzK-charged tRNA (GYU) to specifically decode the AXC codon, allowing the production of site-specifically AzK-modified protein product (purple chain with red shape) in concert with the native translational machinery. c Schematic showing an example AzK- IL-2 precursor molecule (purple chain with AzK incorporated). Reaction of the AzK-containing IL-2 proteins with DBCO-mPEG generates the site-specific and covalent attachment of the mPEG moiety to the AzK (one regioisomeric species shown). An array of different IL-2 variants with AzK incorporated into distinct sites was created and the scheme shown was employed to pegylate each variant.
