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  3. Tracking endogenous proteins based on RNA editing-mediated genetic code expansion

Tracking endogenous proteins based on RNA editing-mediated genetic code expansion

  • Nat Chem Biol. 2024 Feb 1. doi: 10.1038/s41589-023-01533-w.
Min Hao # 1,  Xinyu Ling # 1 2,  Yi Sun # 1,  Xue Wang 1,  Wenzhe Li 1,  Liying Chang 1,  Zhiying Zeng 1,  Xiaomeng Shi 1,  Mengxiao Niu 3,  Liangyi Chen 3,  Tao Liu 4
Affiliations

Affiliations

  • 1 State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China.
  • 2 Department of Genetics, Yale University School of Medicine, New Haven, CT, USA.
  • 3 State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, College of Future Technology, Peking University, Beijing, China.
  • 4 State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, Pharmaceutical Sciences, Peking University, Beijing, China. taoliupku@pku.edu.cn.
  • # Contributed equally.
PMID: 38302606 DOI: 10.1038/s41589-023-01533-w
Abstract

Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical Amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

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