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<p>SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here, we introduce improved labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 10<sup>7</sup> s<sup>−1</sup> M<sup>−1</sup>, a 100-fold improvement over the corresponding SNAP-tag–substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a fivefold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translate into greatly improved performance in various live-cell (super-resolution) imaging applications.</p><p></p>

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SNAP-tag2 for faster and brighter protein labeling

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年7月3日

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  • Stefanie Kühn,
  • Veselin Nasufovic,
  • Jonas Wilhelm,
  • Julian Kompa,
  • Eline M. F. de Lange,
  • Yin-Hsi Lin,
  • Cornelia Egoldt,
  • Jonas Fischer,
  • Artem Lennoi,
  • Miroslaw Tarnawski,
  • Jochen Reinstein,
  • Rifka Vlijm,
  • Julien Hiblot,
  • Kai Johnsson

摘要

SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here, we introduce improved labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 107 s−1 M−1, a 100-fold improvement over the corresponding SNAP-tag–substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a fivefold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translate into greatly improved performance in various live-cell (super-resolution) imaging applications.