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.

Figure 1.

Principle of the ThT assay. ThT is added to nucleic acid structures preformed in 50 mM Tris/HCl, pH 7.5, and 50 mM KCl. ThT binds specifically to G4, and its fluorescence is enhanced. In contrast, when the oligonucleotide is single- or double-stranded, a lower fluorescence increase is observed.

Figure 2.

Fluorescence emission spectra (a.u.) of ThT in the presence of various oligonucleotides. Fluorescence emission of ThT alone is shown in black. Fluorescence in the presence of 10 different oligonucleotides is also shown. Samples were prepared at 1 µM strand in 50 mM Tris/HCl, pH 7.5, 50 mM KCl, and ThT was added to a concentration of 0.5 µM.

Figure 3.

Bar graph and dot plot of fluorescence enhancement of ThT in the presence of a variety of nucleic acid structures. Oligonucleotides are described in Table 1. (A) Each bar of the graph corresponds to the fluorescence enhancement in the presence of the indicated sequence. Error bars correspond to S.D. (B) Each point corresponds to fluorescence enhancement in the presence of a different oligonucleotide. The change in fluorescence emission is plotted for DNA and RNA quadruplexes on the left (in blue) and non-G4 structures on the right. Green dots correspond to trinucleotides, purple dot to parallel-duplex, brown dot to the triplex and red to other nonquadruplex-forming sequences. The difference between these two distributions is highly significant (P < 0.001); however, note that Student’s t-test assumes a normal distribution, which is unproven here (and actually unlikely).

Figure 4.

Evidence for G4 formation for the Tet aptamers. (A) Bar graph of fluorescence enhancement with 1 µM indicated oligonucleotide and 0.5 µM ThT. (B) IDS of the four aptamers tested. (C) Representative imino proton spectrum of the four aptamers tested. The presence of peaks between 10.5 and 12 suggests G4 formation.

Figure 5.

Oligonucleotide migration on a nondenaturing gel revealed by three different methods. Two oligonucleotides, a duplex on the left (19AT) and a quadruplex on the right (22Ag), were tested at eight different concentrations (20, 10, 5, 2, 1, 0.5, 0.25 and 0.125 µM). Samples were prepared in a 50 mM Tris/HCl, pH 7.5 buffer with 50 mM KCl and loaded on a nondenaturing 15% acrylamide gel supplemented with 10 mM KCl. The gel was electrophoresed at 18°C. Migration markers are single-stranded dT_n. The gel was visualized by (A) UV shadowing, (B) staining with 0.5 µM of ThT and (C) staining with SYBR Gold.

Figure 6.

G4 structures detected on nondenaturing polyacrylamide gel electrophoresis by ThT staining. Eighteen different oligonucleotides were tested at 2 µM concentration. Samples were prepared in a 50 mM Tris/HCl, pH 7.5, 50 mM KCl and loaded on a nondenaturing 15% acrylamide gel supplemented with 10 mM KCl. The gel was electrophoresed at 20°C. Migration markers were single-stranded dT_n. The gel was visualized by (Top) staining with 0.5 µM of ThT and (Bottom) staining with SYBR Gold. Preferential staining of G4 structures by ThT is observed (upper panel), whereas SYBR Gold reveals all oligonucleotides (duplexes, single strands and quadruplexes).