A Highly Sensitive Water-Soluble Donor-Acceptor Dye for Early-Stage Amyloid Aggregation Kinetics

Area of Science:

• Biochemistry
• Biophysics
• Materials Science

Background:

• Protein aggregation into amyloid fibrils is implicated in various human diseases.
• The standard fluorescent probe, Thioflavin T (ThT), has limitations in detecting early-stage aggregates and certain fibril structures.

Purpose of the Study:

• To introduce and characterize DANIR-2b(2OH), a novel water-soluble fluorescent dye designed to overcome ThT limitations.
• To assess DANIR-2b(2OH)'s efficacy in detecting and tracking amyloid protein aggregation.

Main Methods:

• Synthesis and characterization of DANIR-2b(2OH) photophysical properties.
• Fluorescent spectroscopy (steady-state and time-resolved) to study protein-dye interactions.
• Time-resolved cryo-electron microscopy to confirm aggregate detection.

Main Results:

• DANIR-2b(2OH) successfully binds and detects early prefibrillar aggregates and small fibrils of human Islet Amyloid Polypeptide, which are missed by ThT.
• The dye also tracks aggregation of other amyloid proteins like insulin and Aβ_1-42.
• DANIR-2b(2OH) exhibits environment-sensitive emission, high photostability, and low noise in plate-reader assays.

Conclusions:

• DANIR-2b(2OH) is a sensitive and broadly applicable fluorescent probe for real-time amyloid aggregation measurements and imaging.
• It overcomes key limitations of ThT, enabling detection of previously undetectable aggregation states.
• This advancement facilitates a more comprehensive understanding of amyloid-related disease mechanisms.