High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors, the N³-Modified Thioflavin T Derivatives

Abstract

Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, for these methods, improved rapidity and simplicity have been required for relevant techniques in laboratory research and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N 3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N 3-(d-desthiobiotinyl-PEGylated) thioflavin T (ThT-PD) and N 3-(cortisolyl-PEGylated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. Compared to those of the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with the target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N 3-ThT derivatives by the rapid specific staining of glucocorticoid receptors in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took ∼24 h for immunostaining to capture the corresponding fluorescence images.

Subtle Structural Changes of Dyes Lead to Distinctly Different Fluorescent Behaviors in Cellular Context: The Role of G-Quadruplex DNA Interaction Using Coumarin–Quinazolinone Conjugates as a Case Study

“…To verify this hypothesis, we first carried out a ThT displacement assay in vitro to investigate the fluorescence change. ThT is a versatile fluorescent probe, which has been widely used in G4 DNA and RNA visualization due to their distinguished fluorescence enhancement with G4s. − As shown in Figure S21, CQ treated with KSS in K + gave a strong emission at 510 nm. Upon gradual addition of ThT, the fluorescence peak was shifted to 495 nm (emission of ThT-G4 complex), indicating that ThT can displace CQ from G4s.…”

Effects of Modifying Thioflavin T at the N3-Position on Its G4 Binding and Fluorescence Emission

“…We then demonstrated that ThT-HE binds to parallel G4 in particular and exhibits strong fluorescence while maintaining the low background emission properties of ThT [17]. In addition, we reported the application of ThT-HE to G4 detection systems [18][19][20]. Since then, ThT with an ethyl group introduced at the N 3 -position (ThT-E) has been reported by Guan et al [21], but the derivatization of ThT at the N 3 -position has not seen significant progress otherwise.…”

“…The synthesis of ThT-HE is described in [17] and those of ThT-AE and ThT-OE11 are described in [18]. The syntheses of ThT-OE2 and ThT-SP are described in the supplementary materials.…”