Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand-DNA complexes: the interaction between DNA and calichearubicin B

Abstract

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (phi) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand-DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin gamma1I. The unwinding angle for CRB calculated by this method is -5. 3 +/- 0.5 degrees. Its Ka value is 0.20 x 10(6) M-1. For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds. Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA. The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Figure 1

Schematic Topo I relaxation reactions of ccDNA at no, low ([I]_L), medium ([I]_M), and high ([I]_H) ligand binding. a, Topo I relaxation; b, ligand removal by organic extraction. Ligand molecules are represented as nodules along the helical axis of the ccDNA shown. Reaction A lacks ligand, so the ccDNA product is fully relaxed with ΔLk = 0. In reaction B, enough ligand is bound to titrate out 2 superhelical turns (sht) so that the ccDNA now contains −3 sht. After a and b, these 2 titrated sht are manifested in a final ΔLk of −2. Reaction C depicts just enough bound ligand to fully titrate all 5 sht, so product and starting ccDNAs are indistinguishable. Finally, D shows high-ligand binding to +2 sht beyond the equivalence point of C. In this example, the ccDNA product bears this additional superhelicity in its ΔLk of −7.

Figure 2

Example calculation of φ_ap. Photos of EB-stained gels are shown above with their corresponding Boltzman distributions below. The results shown are from reactions containing (Left) (•) and lacking (Right) (○) the unwinding ligand DAUN, and values for ΔLk as well as electrophoretic dimensions are indicated on the gels. ΔLk_c is −0.951 for the ligand-containing reaction, and the maximum of the ligand-free control reaction, ΔLk°_c, is +1.49. This implies a ΔΔLk_c of −2.44. With N_L = 1.46 × 10¹⁴ ligand per reaction and N_D = 1.17 × 10¹² ccDNA per reaction, Eq. 6 gives φ_ap = −7.03°. This becomes one point in a binding isotherm as shown in Fig. 3.

Figure 3

Binding isotherms for CRB (○, n = 1), DA (□, n = 4), and EB (•, n = 2). Unwinding data are plotted according to Eq. 5. Interception of the ordinate at extrapolated infinite C_N yields φ directly, and the negative inverse of the slope yields K_a. An additional hypothetical isotherm for DA (▪, n = 6) has been included to show that the intercept value of φ does not depend on the value of n used.

Figure 4

Structures of DA, MGOS, and CRB. Note that the connectivity in CRB is through the β-anomer of B.