Fluorescence Imaging of Azobenzene Photoswitching In Vivo

摘要

The photoisomerization of azobenzene has been used to control a wide variety of molecular processes It has been applied to the photocontrol of biomolecular targets (peptides, proteins, and nucleic acids) in vitro and in cell extracts. Recently it has been applied to the photocontrol of coiled-coil proteins in living cells in culture and to the photocontrol of ion channels in vivo. These studies highlight the promise of azobenzene-modified biomolecules as general agents for the remote control of biomolecular function using light. In order to function as agents for controlling molecular events in complex living systems, however, the azobenzene-based photoswitches must be chemically stable in a variety of intracellular environments. At least for certain azobenzene photoswitches used for conformational control, glutathione in the intracellular environment can reduce and inactivate the photoswitch. Enzyme-mediated reduction or other modification, as occurs with numerous azo dyes, is also possible. Even if not inactivated, azobenzene photoswitches may exhibit different isomerization rates in a cellular environment. For instance glutathione has been found to catalyze thermal cis-to-trans isomerization of certain azobenzenes. Fluorescence imaging of azobenzene photoswitching would enable a direct test of the feasibility of using azobenzene for intracellular photocontrol in a living organism. Since azoben-zenes used for conformational control are not intrinsically fluorescent, we developed a fluorescence reporter for azobenzene photoswitching.