Thioflavin T as a fluorescence light-up probe for G4 formation

Abstract

Thioflavin T (ThT) becomes fluorescent in the presence of the G-quadruplex structure such as that formed by the human telomeric motif. In this report, we extend and generalize these observations and show that this dye may be used as a convenient and specific quadruplex probe. In the presence of most, but not all, G4-forming sequences, we observed a large increase in ThT fluorescence emission, whereas the presence of control duplexes and single strands had a more limited effect on emission. This differential behavior allowed us to design a high-throughput assay to detect G4 formation. Hundreds of different oligonucleotides may be tested in parallel for G4 formation with a simple fluorescence plate reader. We applied this technique to a family of aptamers not previously recognized as G4-forming sequences and demonstrated that ThT fluorescence signal may be used to predict G4 formation.

Orienting Tetramolecular G‐Quadruplex Formation: The Quest for the Elusive RNA Antiparallel Quadruplex

“…This significant increase was similar to the well-established c-myc quadruplex structure used as a control in this assay, which is in agreement with the presence of a quadruplex structure in the 6 G_0 substrate. [25] Once again, no significant difference was found between the two cooling procedures. The solution of 6 GT_0 exhibited a very low increase, which possibly supports the presence of some duplex W-C structures but very likely the absence of a quadruplex motif.…”

“…This indicates that duplex formation per se was not sufficient to obtain a stable higher order structure involving four strands. A thioflavin T assay, which consists in the addition of a specific fluorescent G4-dye to a DNA sample, [24,25] showed a remarkable enhancement of the fluorescence emission upon addition of the 6 G_0 structure ( Figure S1 in the Supporting Information). This significant increase was similar to the well-established c-myc quadruplex structure used as a control in this assay, which is in agreement with the presence of a quadruplex structure in the 6 G_0 substrate.…”

“…T3516) and used without further purification. The concentration was calculated by using the molar extinction coefficient at l = 412 nm in water of 36 000 m À1 cm À1 [25] .…”

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

“…59 At present, the G-quadruplex specific ThT has attracted increasing interests, and it has been used as a light-up fluorescence probe for the sensing of the G-quadruplex, biothiols, Hg 2+ , K + , liver-related short gene, and the structure change of i-moif. [60][61][62][63][64][65] Inspired by these studies, we constructed a new FRET system using CP as an donor and ThT as an acceptor, and designed a simple, label-free, and sensitive biosensor for the detection of thrombin in buffer and in diluted serum. An improved sensitivity for thrombin detection was obtained when compared with two recent strategies based on the FRET from the CP to the dye.…”

Label-free Detection of Zn2+ Based on G-quadruplex

“…The binding to most G4s could bring out greatly enhanced emission in the fluorescence intensity of ThT. 45 Recently, by the merit of its excellent property, ThT has been employed as G4s fluorescent indicator for the label-free detection of Tb 3+ , 46 K + , 47 Hg 2+ , 48 biothiols, 49 cancer gene 50 and protein. 51 As shown in Scheme 1a, the biosensor consists of a G-rich DNA H22 and fluorescence dye ThT.…”