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; Peng R Chen

doi:10.1038/s41586-025-09165-x

RNA codon expansion via programmable pseudouridine editing and decoding

Nature. 2025 Jun 25. doi: 10.1038/s41586-025-09165-x. Online ahead of print.

Authors

Jiangle Liu^#^{1

2

3}, Xueqing Yan^#¹, Hao Wu^#^{1

3}, Ziqin Ji², Ye Shan^{2

3}, Xinyan Wang⁴, Yunfan Ran⁵, Yichen Ma^{1

3}, Caitao Li⁶, Yuchao Zhu^{2

7}, Ruichu Gu^{1

8}, Han Wen^{8

9

10}, Chengqi Yi^{11

12

13

14}, Peng R Chen^{15

16

17}

Affiliations

¹ The National Key Laboratory of Gene Function Studies and Manipulation, School of Life Sciences, Peking University, Beijing, China.
² Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
³ Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
⁴ DP Technology, Beijing, China.
⁵ School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
⁶ School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
⁷ Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China.
⁸ Institute for Advanced Algorithms Research, Shanghai, China.
⁹ Beijing Advanced Center of RNA Biology (BEACON), Peking University, Beijing, China.
¹⁰ AI for Science Institute, Beijing, China.
¹¹ The National Key Laboratory of Gene Function Studies and Manipulation, School of Life Sciences, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
¹² Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
¹³ Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
¹⁴ Beijing Advanced Center of RNA Biology (BEACON), Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
¹⁵ Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. pengchen@pku.edu.cn.
¹⁶ Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. pengchen@pku.edu.cn.
¹⁷ Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing, China. pengchen@pku.edu.cn.

^# Contributed equally.

PMID: 40562925
DOI: 10.1038/s41586-025-09165-x

Abstract

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.