ThT 101: a primer on the use of thioflavin T to investigate amyloid formation, 2017, Malmos et al.

[SNT Gatchaman]

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation
Kirsten Gade Malmos; Luis M. Blancas-Mejia; Benedikt Weber; Johannes Buchner; Marina Ramirez-Alvarado; Hironobu Naiki; Daniel Otzen

Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms.

This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT’s amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.

Link | PDF (Amyloid)

 

[SNT Gatchaman]

When it binds to b-sheet-rich structures such as amyloid fibrils, ThT displays enhanced fluorescence and a characteristic blue shift in the emission spectrum from approximately 510 nm in the free state to 480 nm when bound to amyloid fibrils. In protein-only solutions, the interaction of ThT with amyloid fibrils is highly specific. Neither amorphous aggregates nor soluble proteins in folded, unfolded, or partially folded states enhance ThT fluorescence.

However, ThT also interacts with other biomolecules such as DNA by intercalating between DNA base pairs and (more weakly) by binding to DNA grooves. Polysaccharides such as hyalinous cartilage and certain proteins like acetylcholinesterase also induce the characteristic ThT fluorescence, presumably through the existence of appropriate structural cavities. These properties reduce its usefulness for the identification of amyloid deposits in diagnostic histology compared to Congo red and have spurred the development of ThT derivatives such as Amytracker™ and Proteostat®.

 

[SNT Gatchaman]

Small molecule inhibitors often have structures similar to ThT, allowing them to prevent ThT binding by competitive inhibition or by spectral overlap that quenches ThT signals, rather than inhibiting the fibrillation process itself. Even in the absence of spectral overlap, oxidative products of the small molecules may cause quenching and hence report false positive inhibitors.

Small molecules often included in small molecule libraries like catecholamines (dopamine), polyphenols (resveratrol, EGCG), and flavonoids (quercetin) all oxidize to quinones, which can significantly quench ThT signals. A review on Ab and small molecule inhibitors shows how one would reach false positive conclusions if based only on dye binding assays[109].

[109] is Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid Aβ Self-Assembly (2012, ACS Publications)

Quercetin, resveratrol and EGCG are common supplements taken by patients so there's a possibility that might reduce the apparent amyloid signal.

 

[Jonathan Edwards]

Interesting. I wonder if small molecules may be more of a problem in fluid phase assays. For tissue staining a lot of small molecules may be washed out of the tissue by washing or incubation steps that can be quite lengthy.