Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Developing adaptable biosensors for single-molecule detection is a significant challenge.
• Fluorescent protein-based biosensors offer potential for live-cell imaging but often lack easy customization.
• Existing biosensor platforms may not efficiently target specific subcellular compartments.

Purpose of the Study:

• To create a versatile, single-molecule fluorescent protein-based biosensor platform.
• To enable easy adaptation of biosensors for recognizing diverse molecular targets.
• To validate the biosensor's performance in live human cells and specific organelles.

Main Methods:

• Engineered adaptable, turn-on maturation (ATOM) biosensors by circularly permuting nanobodies and monobodies.
• Inserted these engineered proteins into fluorescent proteins at surface loops.
• Performed multiplexed imaging in live human cells coexpressing different colored ATOM sensors.

Main Results:

• ATOM biosensors demonstrated ligand-dependent chromophore maturation with high turn-on ratios (up to 62-fold in cells, 100-fold in vitro).
• Multiplexed imaging successfully detected specific targets localized to various subcellular compartments.
• Organelle-specific ATOM sensors (ER, mitochondria) accurately detected targeted ligands.

Conclusions:

• The adaptable, turn-on maturation (ATOM) biosensor platform provides a versatile tool for single-molecule detection.
• ATOM biosensors can be rapidly customized and applied to study specific targets in live cells and organelles.
• This technology advances biosensor design for molecular imaging and cellular biology research.