RNA codon expansion via programmable pseudouridylation and codon-specific decoding in mammalian cells
The ability of reprogramming blank codons to encode non-canonical amino acid (ncAA) enables site-specific chemical functionality introduction. However, there are only three endogenous blank codons for translation termination in mammalian cells. To this end, we report a new approach termed RNA Codon Expansion (RCE) to create and reprogram post-transcriptionally modified RNA codons distinguishable with unmodified stop codons. To determine the pseudouridine Codon yields on intented transcripts, we conducted quantitative pseudouridine assessment via bisulfite/sulfite treatment (named "PRAISE"). To evaluate the transcriptome-wide mRNA expression and translatome-wide decoding specificity, we conducted RNA-seq and ribosome profiling.