Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes

Abstract

RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV-Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.

Figure 1. Molecular structure of ThT.

Figure 2. Schematic representations of ThT binding with various RNA forms for G-quadruplex structure recognition.

The fluorescence intensity was substantially enhanced by addition of RNA G-quadruplex sequences, and the ss-RNA and hairpin RNA do not induce enhanced fluorescence.

Figure 3. Fluorescence emission spectra of 2 μM ThT with various oligonucleotides (4 μM) in a 20 mM Tris HCl (40 mM K⁺, pH 7.0) solution.

Figure 4. Dependence of ThT (2 μM) fluorescence intensity at 487 nm for a variety of RNA sequences (4 μM) in a 20 mM Tris HCl (40 mM K⁺, pH 7.0) solution.

Figure 5. Absorption spectra of ThT (2 μM) with RNA G-quadruplex sequence (ADAM10) at eight concentrations (μM): (i) 0, (ii) 0.125, (iii) 0.25, (iv) 0.5, (v) 1, (vi) 2, (vii) 4 and (viii) 8.

Figure 6. Fluorescence intensity enhancement (F/F₀) of ThT (2 μM) at 487 nm plotted as a function of various RNA forms at different concentrations (from 0.125 μM to 8 μM).

The solid lines are the fitted curves assuming 1:1 stoichiometry.

Figure 7. Fluorescence decay traces of ThT (2 μM) in the absence and presence of RNA G-quadruplex structures (4 μM).

The samples were prepared in a 20 mM Tris HCl (40 mM KCl, pH 7.0) buffer solution.

Figure 8

(a) Sequences of non-canonical G-quadruplexes used in this study. G-bases that are marked in blue were involved in forming the RNA G-quadruplex structure. (b) Dependence of the ThT (2 μM) fluorescence intensity in 20 mM Tris HCl buffer (40 mM K⁺, pH 7.0) on the non-canonical G4 sequences and non-G4 sequences (4 μM).

Figure 9. Dependence of the TO and ThT fluorescence intensity on the G4 and non-G4 sequences in a 20 mM Tris HCl (40 mM K⁺, pH 7.0) solution.

(i) tRNA, (ii) ssAf22, (iii) HP18, (iv) tRNA-Ala fragment, (v) tRNA-Cys fragment, (vi) Bulges-TB1 (vii) Spinach, (viii) VEGF and (ix) TRF2.