A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy

• Published in: Nature chemistry Vol. 12; no. 2; pp. 165 - 172
• Main Authors: Wang, Lu, Tran, Mai, D’Este, Elisa, Roberti, Julia, Koch, Birgit, Xue, Lin, Johnsson, Kai
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2020; Springer Nature; Nature Publishing Group
• Subjects: 631/1647/1888/1493; 631/1647/328/2238; 639/638/11/942; 639/638/92/96; Analytical Chemistry; Biochemistry; Carboxyl group; Cell Line, Tumor; Cell Membrane - metabolism; Cell Membrane Permeability; Cell permeability; Chemical compounds; Chemistry; Chemistry and Materials Science; Chemistry, Multidisciplinary; Chemistry/Food Science; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent indicators; Fluorophores; Humans; Inorganic Chemistry; Microscopy, Confocal; Microscopy, Fluorescence; Organic Chemistry; Permeability; Physical Chemistry; Physical Sciences; Probes; Rhodamines - chemical synthesis; Rhodamines - chemistry; Science & Technology; Strategy; Zwitterions; DESIGN; FLUOROPHORES; DYES; LIVE-CELL; RHODAMINE; STED MICROSCOPY; FUSION PROTEINS
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/s41557-019-0371-1

Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy. It is difficult to develop suitable fluorescent probes for live-cell nanoscopy, but a general strategy is now reported that can transform regular fluorophores into fluorogenic probes with excellent cell permeability and low unspecific background signals. Using this approach, probes in a variety of colours were developed for different cellular targets and used for wash-free, multicolour, live-cell confocal and STED microscopy.