Miniature Photoenzyme Enables Organelle-Specific Cellular Control via Deboronative Hydroxylation

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Artificial photoenzymes offer potential for in vitro biocatalysis but face challenges in live-cell applications due to cellular perturbation and oxygen requirements.
• Developing tools for precise, light-activated chemistry within live cells is crucial for understanding and manipulating biological processes.

Purpose of the Study:

• To present miniSOG, a miniature photoenzyme for bioorthogonal deboronative hydroxylation in live cells.
• To demonstrate the versatility and spatiotemporal control of miniSOG in manipulating cellular pathways.
• To establish a genetically encoded platform for targeted cellular modulation with minimal off-target effects.

Main Methods:

• Utilized miniSOG, a 12 kDa photoenzyme, for blue light-activated generation of superoxide radical anion (O2•−).
• Demonstrated miniSOG's ability to photoactivate diverse organoboronates via a unified O2•−-mediated mechanism.
• Showcased spatiotemporally precise photocatalysis in live cells, including organelle-specific targeting and confined reactivity.

Main Results:

• miniSOG enabled bioorthogonal deboronative hydroxylation of 27 diverse organoboronates.
• Achieved precise, light-gated cellular modulation, including mitochondrial depolarization and nuclear epigenetic modification (m6A methylation).
• miniSOG's compact size and localized O2•− diffusion (∼0.2 µm) ensured minimal cellular perturbation.

Conclusions:

• miniSOG is a versatile, genetically encoded photoenzyme platform for precise, light-controlled manipulation of cellular pathways.
• The O2•−-mediated deboronative hydroxylation mechanism offers broad applicability for activating various substrates.
• This technology facilitates targeted cellular interventions with high spatiotemporal resolution and minimal off-target effects.