Genetically encoded alkyne-based cross-linkable probes
Sun, Wei Ni, Yingchun Shu, Xin Liu, Dandan Wu, Haifan Lin, Shixian Zhou, Rong Yang, Bing
Sun, Wei
Ni, Yingchun
Shu, Xin
Liu, Dandan
Wu, Haifan
Lin, Shixian
Zhou, Rong
Yang, Bing
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Issue Date
2026-03-27
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Article
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Keywords
Genetic codon expansion,Rare codon reprogramming,Sortase,Ubiquitin and ubiquitin-like probes,Unnatural amino acids
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Citation
Sun, W., Ni, Y., Shu, X. et al. Genetically Encoded Alkyne-Based Cross-linkable Probes. J. Anal. Test. (2026). https://doi.org/10.1007/s41664-026-00432-y
Abstract
The non-covalent interactions of ubiquitin/ubiquitin-like proteins (Ub/Ubls) with other proteins play a pivotal role in regulating proteins recruitment and activity and ensuring proper protein modifications. However, capturing and identifying transient Ub/Ubl-mediated protein interactions in live cells remains a significant challenge. Genetically encoded cross-linking has emerged as a powerful tool to map such dynamic interactions. Although BprY, a cross-linkable unnatural amino acid (Uaa), has been incorporated into Ub/Ubls for mapping protein-protein interactions (PPIs), its cytotoxicity restricts its practical utility in live cells. Alkyne is non-toxic to cells, and the nucleophilic thiol-alkyne addition reaction facilitates the formation of a covalent thiovinyl bond. This bond can be used for the covalent capture of dynamic and instantaneous PPIs. In this work, we site-specifically incorporate alkyne-based Uaas, 4-propargyloxy-L-phenylalanine (pPR) or enrichable 4-propynyloxy-L-phenylalanine (epPR) into Ub/Ubls. This enables capture and identification of their binding partners in live cells. To extend this technology to polyubiquitin chains, we harness SortaseA-mediated transpeptidation along with Uaas, N6-((2-azidoacetyl)glycyl)-L-lysine (AzGGK), pPR, and epPR to generate cross-linkable branched diubiquitin (diUb). Furthermore, we apply the rare codon recoding (RCR) system to multisite incorporate distinct types of Uaas, epPR, and enrichable fluorosulfate-L-tyrosine (eFSY) into ubiquitin-fold modifier 1 (UFM1) to enhance the efficiency of capturing interacting proteins. © The Nonferrous Metals Society of China 2026.
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Springer Nature
Journal
Journal of Analysis and Testing
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2096241X