Investigating a quadruplex-ligand interaction by unfolding kinetics

Abstract

We have investigated the interaction of the intramolecular human telomeric DNA G-quadruplex with a hemicyanine-peptide ligand, by studying the rate of quadruplex opening with a complementary DNA oligonucleotide. By employing a minimal kinetic model, the relationship between the observed rate of quadruplex opening and the ligand concentration has enabled estimation of the dissociation constant. A van't Hoff analysis revealed the enthalpy and entropy changes of binding to be -77 +/- 22 kJ mol(-1) and -163 +/- 75 J mol(-1) K(-1), respectively. Arrhenius analyses of the rate constants of opening free and bound quadruplex gave activation energies of 118 +/- 2 and 98 +/- 10 kJ mol(-1), respectively. These results indicate that the presence of the ligand has only a small effect on the activation energy, suggesting that the unbinding of the ligand occurs after the transition state for quadruplex unfolding.

Figure 1

FRET-based quadruplex system used in this study. Roman numerals represent the labels given to strands (see Materials and Methods), and TMR and Cy5 are the fluorophores that make up a FRET pair. Quadruplex opening is followed using the change in FRET efficiency between the TMR and Cy5.

Figure 2

Structure of the hemicyanine tetrapeptide conjugate HC.

Figure 3

Scheme used to derive a kinetic model of the opening of quadruplex Q in the presence of ligand L and complementary strand C to give duplex D.

Figure 4

Top: the increase of TMR fluorescence monitored at 580 nm as the quadruplex formed by I:II is opened by III in 10 mM Tris·HCl (pH 7.4) and 100 mM NaCl at 20 °C. Data from runs performed in the presence of ligand HC at concentrations 0 (○), 5 μM (●), and 50 μM (■) are shown, along with single-exponential fits (solid lines). In each series the initial fluorescence intensity has been subtracted from each data point. The rate constants of opening, k_obs, were found to be 0.0086, 0.0053, and 0.0027 s⁻¹ at 0, 5, and 50 μM HC, respectively. Bottom: the variation of observed rate constant with concentration of HC for the hybridization of I:II to III at 20 °C in 10 mM Tris·HCl (pH 7.4) and 100 mM NaCl. The data are shown as open circles and fits to eq 4 as a solid line.

Figure 5

Top: Arrhenius plots of the opening rates of I:II (k₁, ●) and HC·I:II (k₂, ▲), respectively. Bottom: van't Hoff plot of the dissociation constant (K_d, ■) of HC to I:II. Activation energies for k₁ and k₂ obtained from the fits are 118 ± 2 and 98 ± 10 kJ mol⁻¹, respectively, and the thermodynamic parameters found are ΔH = −77 ± 22 kJ mol⁻¹ and ΔS = −163 ± 75 J mol⁻¹ K⁻¹.