DNA binding by the antimalarial compound artemisinin

Abstract

Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.

Figure 1

Secondary structures of the nucleic acid constructs used in this study and chemical structures of ART and 2-deoxyartemisinin. Dashes between nucleotides indicate Watson–Crick base pairs and dots indicate non-Watson–Crick base pairs. For the hybrid DNA/RNA dodecamer the RNA strand is shown in lowercase letters.

Figure 2

ITC thermograms showing interaction of ART with the MN4 aptamer in buffer containing 20 mM TRIS (pH 7.4), 5 mM KCl, 2.4% (v/v) DMSO, in (A) 140 mM NaCl and (B) 0 mM NaCl. (C) Interaction of 2-deoxyartemisinin with the MN4 aptamer in 20 mM TRIS (pH 7.4), 140 mM NaCl, 5 mM KCl, 1.5% (v/v) DMSO. (D) Quinine titrated into the MN4·ART complex. (E) ART titrated into the MN4·quinine complex. Unless otherwise specified, ITC competition experiments were acquired in buffer containing 20 mM TRIS (pH 7.4), 140 mM NaCl, 5 mM KCl, 1% DMSO. All ITC data were acquired at 15 °C.

Figure 3

Native mass spectrometry showing the interaction between ART and the MN4 DNA aptamer forms a non-covalent complex. (A) Native mass spectrum of the MN4 aptamer with ART (1:10 aptamer:ART ratio) in 300 mM ammonium acetate. Theoretical m/z-values of the apo form (dashed lines) and the 1:1 stoichiometry of the complex (dotted lines) are indicated for the 6+ and 5+ charge states. (B) The percentage of observed MN4·ART complex in relation to the total aptamer signal, i.e., unbound and 1:1 bound peaks, plotted in function of the trap collision energy (i.e. acceleration voltage).

Figure 4

(A) ¹H NMR spectra showing the imino proton resonances of MN4 as ART is titrated into the sample up to a 1:1.1 molar ratio of aptamer to ligand. Sample contained 1.4 mM aptamer, 20 mM H_xNa_yPO₄, pH 7.4, 10% D₂O. Final titration point contains ~ 3% DMSO-d₆. Spectra acquired at 5 °C. (B) Histograms showing the absolute value of the difference in peak position of the imino protons for free and ligand-bound MN4. Data is normalized to the largest chemical shift difference value. Displayed ligands are ART (red), quinine (green), cocaine (blue) and amodiaquine (purple). The cocaine, amodiaquine and quinine change in chemical shift data are based on previous published data^,.

Figure 5

Analysis of the thermal stability of MN19 free and ligand-bound using UV melting plots. Shown in (A) are the normalized average of UV absorbance values at 260 nm versus temperature for the unbound MN19 (black), ART-bound MN19 (blue), and quinine-bound MN19 (red) as a positive control. Displayed in (B) are the first derivative plots. Dashed lines indicate the obtained T_m points of the aptamer. Data acquired in PBS (pH 7.4), 3% (v/v) acetonitrile. Each data point denotes an average of three experiments with the error ribbons corresponding to one standard deviation.

Figure 6

Detection of ART using the photochrome aptamer switch assay. Panel (A) displays fluorescence decay plots of 0.1 µM unbound MN19-SITS (black) and as a function of zero to 4.2 µM ART concentration (light blue). The MN19-SITS is continuously excited at 340 nm and the emission at 422 nm are simultaneously detected as a function of time. Each normalized decay plot is fitted to the first-order decay function (solid lines) to quantify the apparent trans–cis decay kinetics (Eq. 1; k_app). Panel (B) calibration plot for the normalized average k_app values of MN19-SITS against ART concentrations. The concentration limit of detection (C_LoD) obtained is (0.22 ± 0.02) µM. Triplicated experiments were performed in 20 mM Tris (pH 7.4), 140 mM NaCl at 20 °C. The error bars correspond to one standard deviation.