Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3

Abstract

Specific guanine rich nucleic acid sequences can form non-canonical structures, like the four stranded G-quadruplex (GQ). We studied the GQ-forming sequence (named HepB) found in the genome of the hepatitis B virus. Fluorescence-, infrared- and CD-spectroscopy were used. HepB shows a hybrid form in presence of K⁺, but Na⁺, Li⁺, and Rb⁺ induce parallel structure. Higher concentrations of metal ions increase the unfolding temperature, which was explained by a short thermodynamic calculation. Temperature stability of the GQ structure was determined for all these ions. Na⁺ has stronger stabilizing effect on HepB than K⁺, which is highly unusual. The transition temperatures were 56.6, 53.8, 58.5 and 54.4 °C for Na⁺, K⁺, Li⁺, and Rb⁺ respectively. Binding constants for Na⁺ and K⁺ were 10.2 mM and 7.1 mM respectively. Study of three ligands designed in cancer research for GQ targeting (TMPyP4, BRACO19 and PhenDC3) showed unequivocally their binding to HepB. Binding was proven by the increased stability of the bound form. The stabilization was higher than 20 °C for TMPyP4 and PhenDC3, while it was considerably lower for BRACO19. These results might have medical importance in the fight against the hepatitis B virus.

Figure 1

(a) Fluorescence spectra of HepB labeled by a FRET pair of FAM and TAMRA in K-phosphate buffer containing 140 mM K⁺ ion at few selected temperatures. (b) The donor fluorescence intensity vs. temperature. The line represents the fitting of Eq. (6).

Figure 2

(a) Fluorescence spectra of HepB_FRET in presence of Na⁺, K⁺, Li⁺ and Rb⁺ at 30 °C. These spectra are normalized to the donor intensity around 518 nm. (b) CD spectra of HepB at room temperature in presence of the same ions.

Figure 3

Infrared spectra of HepB in presence of 100 mM Na⁺, K⁺, Li⁺ and Rb⁺ at pD7.4 at 30 °C.

Figure 4

The unfolding temperature of HepB versus concentration of Na⁺ (filled circle) and K⁺ (filled square) ions. The fitted curves corresponding to the Eq. (10) are also plotted.

Figure 5

Relative donor fluorescence intensity of HepB as function of metal ion concentrations for Na⁺ (filled circle) and K⁺ (filled diamond) at 30 °C. Solid lines show the fitting to Eqs. (2) and (4).

Figure 6

Absorbance spectra of TMPyP4 (0.5 μM) and HepB at concentrations from 10 nM to 10 μM. In 100 mM K-phosphate buffer pH 7.4. Arrows show the spectral changes due to increasing HepB concentration. The curves are corrected for the dilution of TMPyP4.

Figure 7

Fluorescence intensity of HepB_FRET (filled square), HepB_FRET + BRACO19 (filled circle), andHepB_FRET + BRACO19 + excess (nonlabeled) HepB (open triangle).