A bioorthogonal 'turn-on' fluorescent probe for tracking mitochondrial nitroxyl formation

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Biology

Background:

• Mitochondrial reactive nitrogen species (RNS) play crucial roles in cellular signaling and disease.
• Accurate detection of specific RNS like nitroxyl (HNO) in live cells is challenging.
• Existing methods may lack specificity or sensitivity for endogenous HNO.

Purpose of the Study:

• To develop a novel fluorescent probe for sensitive and specific detection of mitochondrial HNO.
• To enable real-time imaging of HNO formation in live cells.
• To investigate the role of mitochondrial HNO in cellular processes.

Main Methods:

• Synthesis and characterization of a bioreductant-resistant 'turn-on' fluorescent probe, Mito-1.
• In vitro and in cellulo validation of Mito-1's response to HNO.
• Confocal and two-photon microscopy for live-cell imaging of mitochondrial HNO.
• Quantification of HNO detection limits using Mito-1.

Main Results:

• Mito-1 exhibits high sensitivity for HNO detection, with a limit as low as approximately 18 nM.
• The probe demonstrates bioreductant resistance, ensuring stability in cellular environments.
• Mito-1 successfully detected both exogenous and endogenous mitochondrial HNO in live cells.
• Two-photon imaging capabilities allowed for noninvasive visualization of mitochondrial HNO.

Conclusions:

• Mito-1 is a robust and sensitive fluorescent probe for detecting mitochondrial HNO in live cells.
• The probe facilitates the study of HNO's role in cellular milieus.
• Mito-1 offers a valuable tool for noninvasive imaging of mitochondrial nitroxyl dynamics.