Green monomeric photosensitizing fluorescent protein for photo-inducible protein inactivation and cell ablation

Area of Science:

• Biochemistry
• Molecular Biology
• Optogenetics

Background:

• Photosensitizing fluorescent proteins generate reactive oxygen species (ROS) upon light irradiation, enabling spatiotemporal control of biological processes.
• Existing photosensitizers offer limited color options, restricting multi-spatiotemporal control of inactivation.
• Developing new photosensitizing proteins with distinct spectral properties is crucial for expanding the optogenetic toolbox.

Purpose of the Study:

• To develop a novel photosensitizing fluorescent protein, SuperNova Green, to expand the available color palette for optogenetic applications.
• To characterize the ROS generation properties of SuperNova Green.
• To demonstrate the utility of SuperNova Green for spatiotemporal protein inactivation and cancer cell ablation.

Main Methods:

• Development of SuperNova Green from a red fluorescent protein precursor.
• Characterization of ROS production, including singlet oxygen and superoxide, upon blue light irradiation.
• Application of SuperNova Green for targeted inactivation of phospholipase C-δ1 and in vitro cancer cell ablation.
• Demonstration of multi-spatiotemporal control using SuperNova Green in conjunction with its red variant, SuperNova.

Main Results:

• SuperNova Green effectively generates ROS, primarily superoxide and its derivatives, upon blue light excitation.
• Specific inactivation of the pleckstrin homology domain of phospholipase C-δ1 was achieved using SuperNova Green.
• In vitro ablation of cancer cells was successfully demonstrated.
• Independent and spatiotemporal control of protein inactivation and cell ablation was achieved by combining SuperNova Green with SuperNova.

Conclusions:

• SuperNova Green represents a valuable addition to the photosensitizing protein toolbox.
• This development enhances optogenetic control over protein inactivation and cell ablation.
• The availability of multiple colors, like SuperNova Green and SuperNova, enables sophisticated multi-spatiotemporal optogenetic experiments.