| Autor: |
Yang S; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Zou LH; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Li R; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Jiang Y; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Ren F; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China., Shao A; School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China. |
| Abstrakt: |
Photoactivatable fluorescence imaging is one of the most valuable methods for visualizing protein localization, trafficking, and interactions. Here, we designed four bioorthogonal fluorescent probes K1 - K4 by installing photoactive cages and HaloTag ligands onto the different positions of the coumarin fluorophore. Although K1 - K4 all exhibited rapid photostimulated responses in aqueous solution, only K3 was found to have an obvious aggregation-induced emission (AIE). Next, macromolecular fluorescent probes K n =1/2/3/4 _ POIs were obtained by covalently attaching K1 - K4 to HaloTag-fused proteins of interest (POIs). K n =3/4 _ POIs exhibited a higher fluorescence increase than that of K n =1/2 _ POIs upon photoactivation in both liquid and solid phases. Moreover, K3 _ GFP _ Halo and K4 _ GFP _ Halo presented the fluorescence resonance energy transfer (FRET) from photocleaved K3 and K4 to GFP in the protein complex. We further examined the fluorescence labeling ability of K1 - K4 to intracellular IRE1_Halo protein and found that K3 and K4 containing the HaloTag ligand on the C4 position of coumarin could be retained in cells for long-term tracking of the IRE1_Halo protein. Hence, we established a platform of novel bioorthogonal fluorescent probes conjugating onto Halo-tagged POIs for rapid photoactivation in vitro and in cells. |
