Targeting DNA junction sites by bis-intercalators induces topological changes with potent antitumor effects

Abstract Targeting inter-duplex junctions in catenated DNA with bidirectional bis-intercalators is a potential strategy for enhancing anticancer effects. In this study, we used d(CGTATACG)2, which forms a tetraplex base-pair junction that resembles the DNA–DNA contact structure, as a model target for two alkyl-linked diaminoacridine bis-intercalators, DA4 and DA5. Cross-linking of the junction site by the bis-intercalators induced substantial structural changes in the DNA, transforming it from a B-form helical end-to-end junction to an over-wounded side-by-side inter-duplex conformation with A-DNA characteristics and curvature. These structural perturbations facilitated the angled intercalation of DA4 and DA5 with propeller geometry into two adjacent duplexes. The addition of a single carbon to the DA5 linker caused a bend that aligned its chromophores with CpG sites, enabling continuous stacking and specific water-mediated interactions at the inter-duplex contacts. Furthermore, we have shown that the different topological changes induced by DA4 and DA5 lead to the inhibition of topoisomerase 2 activities, which may account for their antitumor effects. Thus, this study lays the foundations for bis-intercalators targeting biologically relevant DNA-DNA contact structures for anticancer drug development.


A. Supplementary Note
General procedure for compound synthesis: Commercially available chemicals, including 9chloroacridine, 1,5-diaminopentane, and 1,4-diaminobutane, were purchased from Sigma-Aldrich or TCI and used as received.All reagents were analytical grade and were used without further purification.Ethanol (EtOH) was distilled under nitrogen, using CaH2 as a drying agent, and stored in N2-filled reservoirs with four molecular sieves before use. 1 H nuclear magnetic resonance (NMR) spectra were collected on a Bruker Avance 300 spectrometer.Chemical shifts for 1 H spectra were recorded in ppm relative to the residual proton (1H dimethyl sulfoxide [DMSO]-d6: δ 2.50).Density functional theory calculations were performed using the Gaussian 09 program.Geometry optimizations were performed with the B3LYP functional and 6-31G* basis sets.The solvation free energy was investigated using the self-consistent reaction field and solvation model (1).Elemental analysis and mass spectrometry analysis were performed on a Heraeus CHN-OS Rapid Elemental Analyzer and a JEOL JMX-SX/SX 102A Mass Spectrometer, respectively, at the Instruments Center of National Chung Hsing University, Taiwan.

Figure S1 :
Figure S1: Crystal structure d(CGTATACG)2 DNA alone.(A) 2Fo-Fc electron density maps of the refined structure of d(CGTATACG)2 DNA duplex in an asymmetric unit contoured at 1.0 σ level.(B) Comparison of two adjacent duplexes in the crystal symmetry shows identical duplexes view from the side and top.The superposition of these duplexes shows a root mean square (R.M.S.) deviation of 0.001 Å.

Figure S2 :
Figure S2: Analysis of binding stoichiometry in solution of DA4 and DA5 with d(CGTATACG)2.Jobtype titration plot for DA4 and DA5 in a buffer containing 50 mM sodium cacodylate trihydrate (pH 7.3) and 5 mM magnesium chloride hexahydrate at 25°C.The total concentration of compounds (DA4 or DA5) and DNA was set at 100 μM.The plot at 272 nm shows a distinctive maximum at about 0.66 molar fraction of compounds, indicating approximately two molecules of DA4 or DA5 bind to a duplex DNA.

Figure
Figure S3: 2Fo-Fc electron density maps of the refined structures of DA4-DNA and DA5-DNA complexes contoured at 1.0 σ level.The d(CGTATACG)2 DNA is complexed with (A) DA4 (DA4-DNA complex) and (B) DA5 (DA5-DNA complex).DNA backbones are represented in yellow and blue sticks while compounds are shown in pink stick representation.Metal ions including manganese (purple), magnesium (green) and waters (cyan) are shown in spheres.

Figure S4 :
Figure S4: Superimposition of DA4-DNA and DA5-DNA complexes.(A) Overlay of two adjacent DNA duplexes (blue and pink colored cartoons) in the DA4-DNA complex (left panel) and DA5-DNA complex (right panel), showing that these duplexes are identical.(B) Superimposition of the upper C1-G16/G2-C15 chromophore binding site of one duplex (blue colored sticks) with the C9*-G8*/G10*-C7* site (pink colored sticks) in another duplex showing the differences in interduplex bis-intercalation of DA4 and DA5 structures.

Figure S5 :
Figure S5: Correlation between the quality of the electron density map and the atomic models of DA4 and DA5 in two crystal structure complexes.Shown is the refined 2mFo-DFc maximum likelihoodweighted Fourier electron density map in ccp4 format, countered at the 1.0 σ level with a 2.0 Å carve radius (light blue mesh).The mFo-DFc difference map (red mesh at 3.0 σ) shows a good fit of the geometries of DA4 (orange sticks) and DA5 (green sticks) in the two crystal structures.

Figure S6 :
Figure S6: Stabilizing effects of DA4 and DA5 on d(CGTATACG)2 DNA analyzed by circular dichroism (CD) and melting temperature (Tm).(A) CD spectra of d(CGTATACG) in the presence of different ratios of DA4 and DA5.Spectra were recorded in the presence of 20 μM oligonucleotides prepared in a buffer containing 20 mM sodium cacodylate (pH 7.3), 100 mM KCl and 5 mM magnesium chloride.(B) Effects of DA4 and DA5 on stability of d(CGTATACG)2 DNA. 3 μM DNA duplex was used to determine the melting temperature (Tm) in absence (black line) and presence (pink and green lines) of ligands at DNA:ligand ratio of 1:4 in the same buffer as the CD experiment.This ratio was chosen as CD spectra exhibited saturation at 278 nm after the addition of four equivalents of DA5.

Figure S7 :
Figure S7: Terminal base pair flipping.Flipping of cytosine (C1 and C9) and guanine (G8 and G16) away from helical axis is shown in DNA alone structure of d(CGTATACG)2.

Figure S9 :
Figure S9: Cytotoxicity of DA4 and DA5 on SW620 and A549 cancer cells.The IC50 values against the proliferation of cancer cells are determined for 48 h of treatment for three independent experiments.Graphs show dose-response curves after treatment of compounds.

Figure S10 :
Figure S10: CD spectra analysis of different DNA oligonucleotide sequences with DA4 and DA5.CD spectra of hairpin-, bulge-, triplex-and quadruplex-containing oligonucleotides (the sequences are shown on the left in the figure) in the presence of different ratios of DA4 and DA5.The spectra show that both DA4 and DA5 can effectively interact with hairpin-and bulge-containing CG-rich sequences and significantly alter DNA conformation, similar to CGTATACG duplex DNA CD spectra.DA4 and DA5 did not show strong alterations in the spectra characteristic of triplex and human-telomeric (HTG22) hybrid G-quadruplex (K + form) forming sequences.Spectra were recorded in presence of 20 μM oligonucleotides prepared in a buffer containing 20 mM sodium cacodylate (pH 7.3), 100 mM KCl and 5 mM magnesium chloride.

Table S1 .
Crystallographic and refinement statistics of the structures presented in this study.