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Publications

Journal Article Ppublish

PubMed: 40680052 DOI: 10.1073/pnas.2426354122

Thioether editing generally increases the photostability of rhodamine dyes on self-labeling tags.

Proc Natl Acad Sci U S A 122(30):e2426354122

Authors: Jing Ling, Yuan Zhang, Yongzhen Hei, Julian Kompa, Chen Yang, Bo Wang, Junwei Zhang, Jiasheng Du, Tatjana Rudi, Kecheng Zhang, Jingfu Sun, Wenjuan Wang, Sebastian Fabritz, Yulong Li, Wulan Deng, Peng Zou, Chunlai Chen, Zhixing Chen

Affiliations

  • Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China.
  • Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, College of Future Technology, Peking University, Beijing 100871, China.
  • State Key Laboratory of Membrane Biology, Beijing Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing 100084, China.
  • Department of Chemical Biology, Max Planck Institute for Medical Research, Heidelberg 69120, Germany.
  • Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Biomedical Pioneering Innovation Center, Peking University, Beijing 100871, China. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China.
  • Mass Spectrometry Core Facility, Max Planck Institute for Medical Research, Heidelberg 69120, Germany.
  • Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, College of Future Technology, Peking University, Beijing 100871, China.
  • Peking University-Nanjing Institute of Translational Medicine, Nanjing 211800, China. Genvivo Biotech, Nanjing 211800, China.
  • School of Life Sciences, Technology Center for Protein Sciences, Tsinghua University, Beijing 100084, China.
  • Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing 100871, China. IDG/McGovern Institute for Brain Research at Peking University, Beijing 100871, China. Chinese Institute for Brain Research, Beijing 102206, China.
  • Biomedical Pioneering Innovation Center, Peking University, Beijing 100871, China.
  • Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China.
  • Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. IDG/McGovern Institute for Brain Research at Peking University, Beijing 100871, China. Chinese Institute for Brain Research, Beijing 102206, China. College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of the Ministry of Education, Peking University, Beijing 100871, China.
  • Peking-Tsinghua Center for Life Science, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, College of Future Technology, Peking University, Beijing 100871, China. Peking University-Nanjing Institute of Translational Medicine, Nanjing 211800, China. Genvivo Biotech, Nanjing 211800, China. State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, Beijing 100871, China. Beijing National Laboratory for Molecular Sciences, Beijing 100190, China.

Abstract

Self-labeling protein tags are widely used in advanced bioimaging where dyes with high-photon budgets outperform their fluorescent protein counterparts. Further increasing the emitted photon numbers of dye-tag systems is actively pursued by both new fluorophore chemistry and protein engineering. By scrutinizing the protein microenvironment of fluorophores, here we propose that proximal thioether groups negatively affect the photostability of the dye-tag system. We attribute the disparity in photostability of rhodamine dyes on HaloTag, SNAP-tag, and TMP-tag3 to the influence of the inherent thioether linkage within the SNAP-tag and TMP-tag3. This photochemical pathway leads us to further devise tags with higher photostability. We first show that rhodamine dyes on TMP-tag3.1, which employs a proximity-induced SuFEx reaction instead of a thiol-acrylamide addition to replace the thioether adduct, achieve photon budgets comparable to those ligands on HaloTag. We further showcase that by mutating the methionine near the fluorophore pocket, HaloTag: M175L generally gives up to four times enhancement on photostability when labeled with red and far-red rhodamines. The enhancement of HaloTag modification is demonstrated with single-molecule fluorescence imaging, live-cell fluorescence imaging, and voltage imaging. During time-lapse imaging, gradual photooxidation of Met leads to a reduced photobleaching rate, mechanistically supporting the thioether pathway hypothesis. Our findings suggest that thioether editing on self-labeling tags is a general strategy to enhance the photostability of fluorophores for advanced time-lapse imaging techniques.

MeSH

Time-Lapse Imaging, Budgets