北京大学陈鹏研究小组取得一项新突破。他们提出了通过可编程伪尿嘧啶编辑和解码的RNA密码子扩展。这一研究成果于2025年6月25日发表在国际顶尖学术期刊《自然》上。

为了产生更多真正的空白密码子，该研究团队开发了一种RNA密码子扩增（RCE）策略，该策略在特定的mRNA转录本上引入并解码生物正交可分配的假二吡啶（Ψ）密码子（ΨGA， ΨAA或ΨAG），以在哺乳动物细胞中整合ncAA。RCE策略包括可编程向导RNA4、工程解码器tRNA和氨基酰基-tRNA合成酶。研究人员首先开发了RCE（ΨGA）系统，该系统通过ΨGA密码子将功能性ncAAs整合到蛋白质中，与遗传密码扩展系统相比，显示出更高的翻译体范围和蛋白质组学特异性。该团队进一步扩展了他们的策略，生产了RCE（ΨAA）和RCE（ΨAG）系统，所有三个Ψ密码子：（Ψ密码子）-tRNA^Pyl对都表现出相互正交性。

此外，课题组证明了RCE系统与双ncAA编码的遗传密码扩展策略兼容。总之，RCE方法利用Ψ作为转录后“字母”编码和解码特定mRNA转录本中的RNA密码子，为真核细胞中基因字母表扩增和位点特异性ncAA结合开辟了新的途径。

研究人员表示，非规范氨基酸（non-canonical amino acids, ncAAs）的掺入使得细胞化化学能够调整蛋白质的功能。遗传密码扩展通过将终止密码子重新分配为“空白”密码子，为ncAA编码提供了一种通用方法；然而，它与细胞转录本的翻译终止并不完全正交。

附：英文原文

Title: RNA codon expansion via programmable pseudouridine editing and decoding

Author: 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.

Issue&Volume: 2025-06-25

Abstract: 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 (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.

DOI: 10.1038/s41586-025-09165-x

Source: https://www.nature.com/articles/s41586-025-09165-x