RNA codon expansion via programmable pseudouridine editing and decoding

• 成果类型: Article
• 署名作者: Liu, Jiangle; Yan, Xueqing; Wu, Hao; Ji, Ziqin; Shan, Ye; Wang, Xinyan; Ran, Yunfan; Ma, Yichen; Li, Caitao; Zhu, Yuchao; Gu, Ruichu; Wen, Han; Yi, Chengqi; Chen, Peng R.
• 署名单位: Peking University; Peking University; Peking University; Shanghai Jiao Tong University; ShanghaiTech University; Peking University; Peking University
• 刊物名称: Nature
• ISSN/ISSBN: 0028-1873
• DOI: 10.1038/s41586-025-09165-x
• 发表日期: 2025-07-31
• 关键词: in-vivo; amber; translation; degradation; EVOLUTION; database; p53

摘要

The incorporation of non-canonical amino acids (ncAAs) enables customized chemistry to tailor protein functions1, 2-3. 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 (Psi) codons (Psi GA, Psi AA or Psi AG) 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(Psi GA) system, which incorporates functional ncAAs into proteins via the Psi GA 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(Psi AA) and RCE(Psi AG) systems, with all three Psi codon:(Psi codon)-tRNAPyl 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 Psi 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.