A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps

Abstract

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.

Figure 1.

Ru-phen-dppz motifs elongate DNA. (A) The large aromatic dipyridophenazine ring of Ru(phen)₂dppz²⁺ (referred to as Rudppz in the text) intercalates into dsDNA. (B) Two Rudppz connected by a flexible linker (the complex referred to as flex-Ru2). (C) Cycles of force extension and release for DNA (black lines) and DNA in the presence of 5 nM of flex-Ru2 (green lines). To elucidate the kinetics of flex-Ru2, the force was fixed at 50 pN, 30 pN and 20 pN (20 pN is shown here in the gray box), while the increasing extension was recorded. (D) Flex-Ru2 intercalation kinetics for increasing ligand concentrations of 1, 3, 5 and 7 nM (purple, blue, green and red) when held at a force of 20 pN. Fits (black) are to the model of Equation (3), and these results are included in Figures 3 and 4.

Figure 2.

DNA saturated with flex-Ru2 (red), from constant force measurements, and fit to Equation (1). Lines for dsDNA (black) taken from Wenner et al. (27) for dsDNA saturated with Rudppz (pink) from Vladescu et al. (4) and fitted data for ssDNA shown (blue). The parameters of the corresponding WLC fits are collected in Table 1.

Figure 3.

Kinetics of flex-Ru2 binding to dsDNA. (A) Measured fast and slow rates versus concentration of flex-Ru2 at three forces: 20, 30 and 50 pN (red, green and blue). Data points are rates k_f and k_s obtained by fitting the length versus time for the flex-Ru2/DNA complex to Equation (3). Lines are the results of fits to Equation (6) (solid lines) and Equation (7) (dotted lines) that determine the elementary rates of the two-step reaction. (B) Fitted values of elementary rates of the two-step intercalation, giving the forward rates k₁ and k₂ (solid purple and orange symbols) and reverse rates k₋₁ and k₋₂ (open purple and orange symbols). Lines represent fits to Equation (8), and give the force independent elementary rates and transition distances, as described in the text. Fitted parameters are shown in Table 2. (C) Force dependent binding constants for each step K_d1 (cyan) and K₂ (gold) and for overall binding K_d (magenta), determined from the elementary rates. Lines denote fits to Equation (10), which give the force independent binding constants and equilibrium length changes, which are included in Table 2.

Figure 4.

Equilibrium analysis of DNA elongations induced by flex-Ru2 intercalation. (A) Measured equilibrium flex-Ru2/DNA length expressed as an occupancy (Θ, relative to the saturated values of Figure 2) as a function of ligand concentration (C) for the forces of 20, 30 and 50 pN (red, green and blue). Fits to Equation (12) (lines) determine the binding constant K_d for each force. (B) The ratio of the fast and slow elongation amplitudes, dx_f/dx_s, as a function of C (20 pN: red, 30 pN: green and 50 pN: blue). Fits of these data points to Equation (15) (lines) determine K_d1(F). (C) Binding constants K_d1 (cyan), K_d (magenta) and K₂ (gold) versus force, as obtained from the fits of the data in Figure 4A and B to Equation (10) (lines), with K₂ calculated as K₂ = K_d1/K_d. Fitted zero-force binding constants and the flex-Ru2/DNA length changes associated with each K are collected in Table 2.

Figure 5.

Zero-force free energy profile of the DNA/flex-Ru2 complex versus elongation. The three free-energy minima on this diagram correspond to the non-intercalated, mono-intercalated and bis-intercalated states illustrated in the figure. At the flex-Ru2 concentration of C = K_d = 15 nM, the free energy of the non-intercalated and bis-intercalated states are the same and are taken here as the zero free energy reference state. At higher C = K_d1 = 35 nM the free energies of the non-intercalated and the mono-intercalated states are the same, and equal to k_BT^.ln(K₂) = k_BT^.ln(35/15) = 0.85 k_BT. Only the non-intercalated state free energy is affected by C, as illustrated by the two lines (solid line for 15 nM and dashed line for 35 nM). The free energy barriers between the local free energy minima were calculated, as discussed in the main text, and all extensions are taken from Table 2.