The article reviews the molecular mechanism of Thioflavin-T (ThT) binding to amyloid fibrils, a widely used fluorescent dye for detecting and studying amyloid structures. ThT's fluorescence emission significantly increases upon binding to amyloid fibrils, making it a powerful tool for both clinical and research applications. The review highlights recent progress in understanding ThT-fibril interactions at an atomic level, which has provided insights into amyloid structures and fibril formation processes. Key findings include: 1. **Spectroscopic Characteristics**: ThT exhibits a dramatic increase in fluorescence upon binding to fibrils, likely due to the immobilization of specific conformers. This immobilization is facilitated by the dye's molecular rotor behavior, where the benzylamine and benzathiole rings rotate freely in solution but are sterically locked in place upon binding to fibrils. 2. **Channel Model**: ThT binds to the cross-β structure of amyloid fibrils, forming a channel along the β-sheet surface. This binding is preferential to aromatic residues, particularly Tyr and Phe, which provide hydrophobic surfaces for ThT binding. 3. **Peptide Self-Assembly Mimics (PSAMs)**: PSAMs, designed to mimic amyloid fibril surfaces, have been used to define the minimal requirements for ThT binding. These studies have shown that a channel formed by aromatic and hydrophobic residues across five or more β-strands is sufficient for high-affinity binding. 4. **Size and Sequence Preferences**: ThT binding sites are typically rich in aromatic residues and require a minimum size of five or more β-strands. Highly charged sequences, such as poly-Lys, do not bind ThT due to electrostatic repulsion. 5. **Non-Fibrillar Structures**: ThT can also bind to hydrophobic pockets in non-fibrillar proteins, which may explain its cross-reactivity in certain cases. 6. **Implications for Amyloid Dyes**: The detailed understanding of ThT binding mechanisms has implications for the design of amyloid dyes and inhibitors, as well as the interpretation of ThT staining results in the presence of other amyloid dyes or inhibitors. The review concludes by emphasizing the importance of further research into the molecular mechanisms of ThT binding to advance the field of amyloid studies and potentially guide the development of new therapies for amyloid-related diseases.The article reviews the molecular mechanism of Thioflavin-T (ThT) binding to amyloid fibrils, a widely used fluorescent dye for detecting and studying amyloid structures. ThT's fluorescence emission significantly increases upon binding to amyloid fibrils, making it a powerful tool for both clinical and research applications. The review highlights recent progress in understanding ThT-fibril interactions at an atomic level, which has provided insights into amyloid structures and fibril formation processes. Key findings include: 1. **Spectroscopic Characteristics**: ThT exhibits a dramatic increase in fluorescence upon binding to fibrils, likely due to the immobilization of specific conformers. This immobilization is facilitated by the dye's molecular rotor behavior, where the benzylamine and benzathiole rings rotate freely in solution but are sterically locked in place upon binding to fibrils. 2. **Channel Model**: ThT binds to the cross-β structure of amyloid fibrils, forming a channel along the β-sheet surface. This binding is preferential to aromatic residues, particularly Tyr and Phe, which provide hydrophobic surfaces for ThT binding. 3. **Peptide Self-Assembly Mimics (PSAMs)**: PSAMs, designed to mimic amyloid fibril surfaces, have been used to define the minimal requirements for ThT binding. These studies have shown that a channel formed by aromatic and hydrophobic residues across five or more β-strands is sufficient for high-affinity binding. 4. **Size and Sequence Preferences**: ThT binding sites are typically rich in aromatic residues and require a minimum size of five or more β-strands. Highly charged sequences, such as poly-Lys, do not bind ThT due to electrostatic repulsion. 5. **Non-Fibrillar Structures**: ThT can also bind to hydrophobic pockets in non-fibrillar proteins, which may explain its cross-reactivity in certain cases. 6. **Implications for Amyloid Dyes**: The detailed understanding of ThT binding mechanisms has implications for the design of amyloid dyes and inhibitors, as well as the interpretation of ThT staining results in the presence of other amyloid dyes or inhibitors. The review concludes by emphasizing the importance of further research into the molecular mechanisms of ThT binding to advance the field of amyloid studies and potentially guide the development of new therapies for amyloid-related diseases.