RNA codon expansion via programmable pseudouridine editing and decoding

The incorporation of non-canonical amino acids (ncAAs) enables customized chemistry to tailor protein functions^1-3. Genetic code expansion offers a general approach for ncAA encoding by reassigning stop codons as the 'blank' codon; however, it is not completely orthogonal to translation termination for cellular transcripts. Here, to generate more bona fide blank codons, we developed an RNA codon-expansion (RCE) strategy that introduces and decodes bioorthogonally assignable pseudouridine (Ψ) codons (ΨGA, ΨAA or ΨAG) on specified mRNA transcripts to incorporate ncAAs in mammalian cells. The RCE strategy comprises a programmable guide RNA⁴, an engineered decoder tRNA, and aminoacyl-tRNA synthetase. We first developed the RCE(ΨGA) system, which incorporates functional ncAAs into proteins via the ΨGA codon, demonstrating a higher translatome-wide and proteomic specificity compared with the genetic code expansion system. We further expanded our strategy to produce the RCE(ΨAA) and RCE(ΨAG) systems, with all three Ψ codon:(Ψ codon)-tRNA^Pyl pairs exhibiting mutual orthogonality. Moreover, we demonstrated that the RCE system cooperates compatibly with the genetic code expansion strategy for dual ncAA encoding. In sum, the RCE method utilized Ψ as a post-transcriptional 'letter' to encode and decode RNA codons in specific mRNA transcripts, opening a new route for genetic alphabet expansion and site-specific ncAA incorporation in eukaryotic cells.