Multiple and Cooperative Binding of Fluorescence Light-up Probe Thioflavin T with Human Telomere DNA G-Quadruplex

Abstract

Thioflavin T (ThT), a typical probe for protein fibrils, also binds human telomeric G-quadruplexes with a fluorescent light-up signal change and high specificity against DNA duplexes. Cell penetration and low cytotoxicity of fibril probes having been widely established, modifying ThT and other fibril probes is an attractive means of generating new G-quadruplex ligands. Thus, elucidating the binding mechanism is important for the design of new drugs and fluorescent probes based on ThT. Here, we investigated the binding mechanism of ThT with several variants of the human telomeric sequence in the presence of monovalent cations. Fluorescence titrations and electrospray ionization mass spectrometry (ESI-MS) analyses demonstrated that each G-quadruplex unit cooperatively binds to several ThT molecules. ThT brightly fluoresces when a single ligand is bound to the G-quadruplex and is quenched as ligand binding stoichiometry increases. Both the light-up signal and the dissociation constants are exquisitely sensitive to the base sequence and to the G-quadruplex structure. These results are crucial for the sensible design and interpretation of G-quadruplex detection assays using fluorescent ligands in general and ThT in particular.

All-Atomic Simulations on Human Telomeric G-Quadruplex DNA Binding with Thioflavin T

“…35−37 Second, ThT has higher binding affinities to the K + promoted hybridized conformations than to the Na + induced antiparallel ("basket") conformer. 35 Third, the disassociation constants as well as the binding stoichiometry of G-quadruplex−ThT binding complexes were investigated. The results suggest that the binding of ThTs on G-quadruplex is a cooperative process and up to four ThTs can bind to one (3 + 1) hybridized Gquadruplex DNA molecule.…”

“…The results suggest that the binding of ThTs on G-quadruplex is a cooperative process and up to four ThTs can bind to one (3 + 1) hybridized Gquadruplex DNA molecule. 35 Additionally, the melting temperature changes of G-quadruplexes upon binding unveil a positive effect of ThT on the stabilization of G-quadruplexes. 36,38 However, the binding mechanism and binding sites of ThT on G-quadruplexes at the atomic level have not been determined yet.…”

Thioflavin T as an Efficient G-Quadruplex Inducer for the Highly Sensitive Detection of Thrombin Using a New Föster Resonance Energy Transfer System

“…48-50 Now, DNA intercalating agents have been used as probes for various DNA structure in addition to DNA-directed therapeutics. 51 Owing to the fact that more than one dye molecule can intercalate into the grooves of a DNA duplex, 52,53 the sensitivity of the sensor would be remarkablely improved by using an intercalating dye and a label-free DNA probe. Studies of our group and 4 fluorescence enhancement (>1700-fold) compared with the enhancement (<250-fold) observed in other single/double strand DNA forms.…”

Exploring the Formation and Recognition of an Important G-Quadruplex in a HIF1α Promoter and Its Transcriptional Inhibition by a Benzo[c]phenanthridine Derivative

“…In H 2 O−CH 3 OH (3:1) with 150 mM NH 4 OAc, an ion at m/z =1323 was observed in the mass spectrum, which is the complex ion [S1 + NH 4 + − 6H + ] 5-(Q1), indicating the formation of a 2-layer Gquadruplex. 28,29 (Figure 1b). In contrast, a mutation of S1, which would completely disrupt the G-quadruplex, only showed a peak at m/z 1320, representing a single S1m without any ammonium ions (Supporting Information Figure S1).…”