We are pioneering the development of bioorthogonal chemistry-based strategies for the precise spatiotemporal control of protein function. By integrating genetic code expansion, computational protein design, and machine learning, we have established a universal "Protein Decaging" platform. This comprehensive framework encompasses a versatile toolkit capable of caging and reactivating a broad spectrum of amino acids (e.g., Lys, Tyr, Glu, Asp) with second-level temporal resolution. Extending beyond chemical innovation, we have developed CAGE-prox and CAGE-Proxvivo technologies to enable programmable gain-of-function studies in complex biological systems. From dissecting signaling kinetics in single cells to modulating therapeutic targets in living animals, our work provides a powerful paradigm for interrogating and manipulating biology in its native context.
Recent representative work:
1) Protein Decaging Toolkit
The development of a protein decaging toolkit provides a universal approach for spatiotemporal control of protein function in living systems. This toolbox has evolved from earlier limitations—such as targeting only a few amino acids like cysteine and serine, and relying on UV photolysis—into a versatile strategy that can be triggered through diverse bioorthogonal and photochemical reactions ( Nat. Chem. Biol. 2016). To date, eight amino acids, including Lys ( Nat. Chem. 2014; Nat. Chem. Biol. 2014), Tyr ( JACS. 2016 ), Glu, Asp (JACS. 2023), Trp ( Nat. Chem. 2024 ), and Phe ( Nat. Chem. In revision）can be site-specifically caged and reactivated, with more amino acids including Thr, Met, Gln, His, and Arg under ongoing evaluation，enabling applications across scales from subcellular organelles to living animals, with time resolution reaching the second level. This suite of tools allows precise modulation of a wide range of protein families, such as kinases, phosphatases, proteases, and readers of post-translational modifications, offering a powerful gain-of-function platform for studying dynamic biological processes in live cells and organisms.
2) Protein Decaging in living cells
A universal gain-of-function approach for selective and temporal control of protein activity in living systems is crucial to understanding dynamic cellular processes. Here we report development of a computationally aided and genetically encoded proximal decaging (hereafter, CAGE-prox) strategy that enables time-resolved activation of a broad range of proteins in living cells and mice. Temporal blockage of protein activity was computationally designed and realized by genetic incorporation of a photo-caged amino acid in proximity to the functional site of the protein, which can be rapidly removed upon decaging, resulting in protein re-activation. We demonstrate the wide applicability of our method on diverse protein families, which enabled orthogonal tuning of cell signalling and immune responses, temporal profiling of proteolytic substrates upon caspase activation as well as the development of protein-based pro-drug therapy. We envision that CAGE-prox will open opportunities for the gain-of-function study of proteins and dynamic biological processes with high precision and temporal resolution ( Nature 2019 ).
3) Decaging in living animals
A universal strategy for precise, on-demand protein activation in living animals is crucial for gain-of-function studies in physiologically relevant settings. CAGE-Proxvivo is a computer-aided proximal decaging platform that enables programmable protein activation and modulation of protein–protein interactions in living mice. By combining machine-learning-guided evolution of aminoacyl-tRNA synthetases with the rational design of proximal "decaging sites," chemically caged amino acids can be site-specifically incorporated to transiently block protein function. Upon administration of a small-molecule trigger, a bioorthogonal cleavage reaction restores protein activity with high precision in vivo. CAGE-Proxvivo shows broad applicability, enabling controlled activation of diverse proteins and cell-type-specific manipulation of physiological phenotypes in living systems. Beyond catalytic-site decaging, it also facilitates precise regulation of protein–protein interactions, exemplified by a "gated" anti-CD3 antibody that allows chemically regulated T-cell recruitment and activation within tumors. Collectively, CAGE-Proxvivo provides a versatile and broadly accessible platform for time-resolved biological studies and on-demand therapeutic modulation in living animals (Cell 2025 ).
Representative publications:
1. Wang, X.; Liu, Y.; Wang, Z.; Zeng, X.; Ngai, W. S. C.; Wang, J.; Zhang, H.; Xie, X.; Zhu, R.; Fan, X.; Wang, C.; Chen P*, Machine-learning-assisted universal protein activation in living mice. Cell 2025, 188 (14), 3696-3714.
2. Zhu Y, Ding W, Chen Y, Shan Y, Liu C,Fan X*, Lin S*, Chen P*, “Genetically encoded bioorthogonal tryptophan decaging in living cells”,Nat.Chem. 2024, 16, 533-42
3. Zhang X, Huang H, Liu Y, Wu Z, Wang F, Fan X*, Chen P*, Wang J*, “Optical control of protein functions via genetically encoded photocaged aspartic acids”, J. Am. Chem. Soc. 2023 ,145, 19218-24.
4. Ngai, W. S. C.; Yang, S.; Zeng, X.; Liu, Y.; Lin, F.; Wang, X.; Zhang, H.; Fan, X.; Chen P*, Bioorthogonally Activatable Base Editing for On-Demand Pyroptosis. J. Am. Chem. Soc. 2022, 144, 12
5. Wang, J.; Wang, X.; Fan, X.; Chen P*, Unleashing the Power of Bond Cleavage Chemistry in Living Systems. ACS Cent. Sci. 2021, 7 (6), 929-943.
6. Liu, S.; Lin, C.; Xu, Y.; Luo, H.; Peng, L.; Zeng, X.; Zheng, H.; Chen P*.; Zou, P., A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons. Nat. Chem. 2021, 13 (5), 472-479.
7. Wang, J.; Liu, Y.; Liu, Y.; Zheng, S.; Wang, X.; Zhao, J.; Yang, F.; Zhang, G.; Wang, C.; Chen P*., Time-resolved protein activation by proximal decaging in living systems. Nature 2019, 569 (7757), 509-513.
8. Wang, X.; Liu, Y.; Fan, X.; Wang, J.; Ngai, W. S. C.; Zhang, H.; Li, J.; Zhang, G.; Lin, J.; Chen P*, Copper-Triggered Bioorthogonal Cleavage Reactions for Reversible Protein and Cell Surface Modifications. J. Am. Chem. Soc. 2019, 141 (43), 17133-17141.
9. Yao, Q.; Lin, F.; Fan, X.; Wang, Y.; Liu, Y.; Liu, Z.; Jiang, X.; Chen P*; Gao, Y., Synergistic enzymatic and bioorthogonal reactions for selective prodrug activation in living systems. Nat. Commun. 2018, 9 (3) , 5032.
10. Fan, X. Y.; Li, J.; Chen P*., Bioorthogonal chemistry in living animals. Natl. Sci. Rev. 2017, 4 (3), 300-302.
11. Li, J.; Chen P*, Development and application of bond cleavage reactions in bioorthogonal chemistry. Nat. Chem. Biol. 2016, 12 (3), 129-37.
