Molecular Rotors: What Lies Behind the High Sensitivity of the Thioflavin-T Fluorescent Marker

Abstract

Thioflavin-T (ThT) can bind to amyloid fibrils and is frequently used as a fluorescent marker for in vitro biomedical assays of the potency of inhibitors for amyloid-related diseases, such as Alzheimer's disease, Parkinson's disease, and amyloidosis. Upon binding to amyloid fibrils, the steady-state (time-integrated) emission intensity of ThT increases by orders of magnitude. The simplicity of this type of measurement has made ThT a common fluorescent marker in biomedical research over the last 50 years. As a result of the remarkable development in ultrafast spectroscopy measurements, researchers have made substantial progress in understanding the photophysical nature of ThT. Both ab initio quantum-mechanical calculations and experimental evidence have shown that the electronically excited-state surface potential of ThT is composed of two regimes: a locally excited (LE) state and a charge-transfer (CT) state. The electronic wave function of the excited state changes from the initial LE state to the CT state as a result of the rotation around a single C-C bond in the middle of the molecule, which connects the benzothiazole moiety to the dimethylanilino ring. This twisted-internal-CT (TICT) is responsible for the molecular rotor behavior of ThT. This Account discusses several factors that can influence the LE-TICT dynamics of the excited state. Solvent, temperature, and hydrostatic pressure play roles in this process. In the context of biomedical assays, the binding to amyloid fibrils inhibits the internal rotation of the molecular segments and as a result, the electron cannot cross into the nonradiative "dark" CT state. The LE state has high oscillator strength that enables radiative excited-state relaxation to the ground state. This process makes the ThT molecule light up in the presence of amyloid fibrils. In the literature, researchers have suggested several models to explain nonradiative processes. We discuss the advantages and disadvantages of the various nonradiative models while focusing on the model that was initially proposed by Glasbeek and co-workers for auramine-O to be the best suited for ThT. We further discuss the computational fitting of the model for the nonradiative process of ThT.

Valence-bond non-equilibrium solvation model for a twisting monomethine cyanine

“…This is Equation 23. This can be used in conjuction with the timescale formula Equation 1 to yield a timescale for rotation of one of the rotors in a monomethine.…”

CuII Binding to Various Forms of Amyloid‐β Peptides: Are They Friends or Foes?

“…[57,58] A very classic assay relies on the use of Thioflavin-T (ThT), a dye that shows a characteristic redshift in the excitation spectrum and strong enhancement of the fluorescence quantum yield upon binding to fibrillary structures. [59][60][61][62] Amyloid fibrils are characterized by laminated -sheets, the strands of which run perpendicular to the long axis of the fibril. [59,62,63] This amyloid composition produces the characteristic reflections (ca.…”

Morphology‐Specific Inhibition of β‐Amyloid Aggregates by 17β‐Hydroxysteroid Dehydrogenase Type 10

“…[43] Secondly, aggregation protocols to reproducibly promote the formation of these A aggregates are well-established and the resulting aggregates have been extensively characterized using structural imaging methods including atomic-force microscopy (AFM) and electron-microscopy (EM). [32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] In a previous study we have already shown using EM that the presence of the HiLyte Fluor 555 dye at the N-terminus of the A(1-42) peptide (A 555 ) does not affect the aggregation kinetics or the morphology of the aggregates. [34] Indeed, we observed amyloid structures of A 555 with diameters of ~ 4 nm (fibrils) and ~ 22 nm (globules) which are identical to those obtained in the absence of N-terminal functionalization.…”