NovaID: photocatalytic proximity labeling with an engineered HyperNova

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Photocatalytic proximity labeling is crucial for mapping molecular interactions in cells.
• Existing methods often rely on specific cofactors, limiting their application.
• There is a need for versatile, genetically encoded photocatalytic platforms.

Purpose of the Study:

• To repurpose HyperNova, a photosensitizer, for genetically encoded photocatalytic proximity labeling.
• To develop and characterize light-activated variants for orthogonal labeling.
• To establish NovaID as a versatile platform for mapping subcellular environments.

Main Methods:

• Repurposing the HyperNova photosensitizer for proximity labeling.
• Engineering a blue-light-activated variant (HyperNova Green).
• Utilizing biotin-aniline as a substrate for labeling.
• Employing dual-wavelength light activation (560 nm and 460 nm).
• Investigating bioluminescence resonance energy transfer (BRET)-mediated activation with Antares2.

Main Results:

• HyperNova enables 560 nm light-activated proximity labeling in various cellular compartments.
• HyperNova Green allows for blue-light (460 nm) activation.
• Orthogonal labeling is achievable using distinct wavelengths in the same cell.
• BRET-mediated activation provides an illumination-free labeling option.
• The NovaID platform demonstrates versatility and spatiotemporal control.

Conclusions:

• NovaID is a compact and adaptable genetically encoded photocatalytic proximity labeling platform.
• It expands the toolkit for mapping local molecular environments in living cells.
• The platform supports orthogonal and illumination-free labeling strategies.