SNAP-Tag-Based Subcellular Protein Labeling and Fluorescent Imaging with Naphthalimides

Genetically encoded technologies provide methods for specific labeling and imaging of proteins, which is essential for the understanding of their subcellular localization and function. Herein, we employ naphthalimide, an efficient two-photon fluorophore, to develop O6-benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP-tag. The three naphthalimide-BG derivatives, TNI-BG, QNI-BG and ONI-BG are conveniently synthesized through modular "click chemistry" with high yields. All of them show high labeling efficiency with SNAP-tag in solution (1~2 ×103 s-1 M-1) and in bacteria. Among them, ONI-BG shows high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP-tag in mammalian cells. The protein labeled naphthalimides exhibit high two-photon absorption cross section, which indicates the potential application in protein specific two-photon fluorescent imaging, such as two-photon fluorescent lifetime imaging and two-photon multi-color imaging. Therefore, ONI-BG is a versatile tool for tracking of subcellular proteins via multiple fluorescent techniques.