Figure 1.
Schematic diagram of the 5′-flanking region of RLX gene.
Figure 2.
Formation of RLX G-quadruplex.
(A) 1H-NMR titration of G-rich sequence S1 with KCl. Increasing amounts of the K+ were serially added to the DNA solution as indicated on each scan. The titration was carried out at 298 K and used K2HPO4-KH2PO4 (pH = 7.0) as buffer solution; (B) CD spectra of 10 µM S1 in the presence of 100 mM KCl or LiCl (30 mM Tris-HCl, pH = 7.4); (C) ESI mass spectrum of 10 µM S1 in 100 mM NH4Ac (pH = 7.0) and 20% methanol solution.
Figure 3.
Normalized CD melting temperature curves of 10 µM (red circle) and 20 µM (black square) RLX G-quadruplex (S1) at 264 nm in 30 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl.
Figure 4.
Complex of RLX G-quadruplex and berberine.
(A) ESI mass spectrum (100 mM NH4Ac, pH = 7.0) and (B) CD spectrum (100 mM KCl, 30 mM Tris-HCl, pH = 7.4) for the complex of 10 µM RLX G-quadruplex (G4) and 40 µM berberine at a molar ratio 1∶4.
Figure 5.
1H-NMR titration of RLX G-quadruplex (G4) with berberine.
Increasing amounts of the berberine were serially added to the DNA solution as indicated on each scan. The titration was carried out at 298 K and used K2HPO4-KH2PO4 (pH = 7.0) as buffer solution.
Figure 6.
CD Tm of complex of RLX G-quadruplex and berberine.
Normalized CD melting temperature curves at 263.8 nm for 10 µM RLX G-quadruplex (S1) before (black square) and after (red circle) adding 40 µM berberine (100 mM KCl, 30 mM Tris-HCl buffer, pH 7.4).
Table 1.
Thermodynamic parameters of RLX G-quadruplex.
Figure 7.
RLX G-quadruplex structure models presented by software Chimera [41].
(A) Top view (B) side view. (The purple balls stand for potassium ions). The docked results of berberine with RLX G-quadruplex presented by software PMV [42] (C) end stacking (D) groove binding. (Berberine was shown as stick model while the G-quadruplex as molecular surface model).
Figure 8.
Results demonstrated that the formation and stabilization of G-quadruplex induced by berberine could increase RLX promoter activity about 10∼40 folds and in a dose-dependent manner (blue column). Berberine has no effect on RLX promoter activity when the G-rich region mutated (red column). Results are presented three to four separate experiments.