RNA codon expansion via programmable pseudouridine editing and decoding

Jiangle Liu, Xueqing Yan, Hao Wu, Ziqin Ji, Ye Shan, Xinyan Wang, Yunfan Ran, Yichen Ma, Caitao Li, Yuchao Zhu, Ruichu Gu, Han Wen, Chengqi Yi () and Peng R. Chen ()
Additional contact information
Jiangle Liu: Peking University
Xueqing Yan: Peking University
Hao Wu: Peking University
Ziqin Ji: Peking University
Ye Shan: Peking University
Xinyan Wang: DP Technology
Yunfan Ran: Shanghai Jiao Tong University
Yichen Ma: Peking University
Caitao Li: ShanghaiTech University
Yuchao Zhu: Peking University
Ruichu Gu: Peking University
Han Wen: Institute for Advanced Algorithms Research
Chengqi Yi: Peking University
Peng R. Chen: Peking University

Nature, 2025, vol. 643, issue 8074, 1410-1420

Abstract: Abstract The incorporation of non-canonical amino acids (ncAAs) enables customized chemistry to tailor protein functions1–3. 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)-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 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.

Date: 2025
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DOI: 10.1038/s41586-025-09165-x

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