Metallohelices stabilize DNA three-way junctions and induce DNA damage in cancer cells

Abstract

DNA three-way junctions (3WJ) represent one of the simplest supramolecular DNA structures arising as intermediates in homologous recombination in the absence of replication. They are also formed transiently during DNA replication. Here we examine the ability of Fe(II)-based metallohelices to act as DNA 3WJ binders and induce DNA damage in cells. We investigated the interaction of eight pairs of enantiomerically pure Fe(II) metallohelices with four different DNA junctions using biophysical and molecular biology methods. The results show that the metallohelices stabilize all types of tested DNA junctions, with the highest selectivity for the Y-shaped 3WJ and minimal selectivity for the 4WJ. The potential of the best stabilizer of DNA junctions and, at the same time, the most selective 3WJ binder investigated in this work to induce DNA damage was determined in human colon cancer HCT116 cells. These metallohelices proved to be efficient in killing cancer cells and triggering DNA damage that could yield therapeutic benefits.

Graphical Abstract

Figure 1.

Metallohelices 1–8 used in this study.

Figure 2.

(A) Structures and abbreviations of DNA junctions along with abbreviations of the individual strands. (B) ΔT_m values for the fluorescently labeled DNA junctions (0.4 μM) upon addition of 0.8 μM 1–8 and in the presence of increasing concentrations (indicated in the Figure and expressed per nucleotide) of dsDNA. The buffer conditions were 10 mM sodium phosphate (pH 7), 100 mM NaCl and 5 mM MgCl₂.

Figure 3.

ΔT_m values for the fluorescent-labeled DNA 3WJ junctions (0.4 μM) upon addition of 0.8 μM 1–8. The schemes above bar graphs show the positions of nucleotides at the branch point of each junction. The buffer conditions were 10 mM sodium phosphate (pH 7), 100 mM NaCl, and 5 mM MgCl_2.

Figure 4.

Assembly of a 3WJ from the self-complementary 12-mer (sc12) in the presence of Λ-8. The concentrations of oligonucleotides were 5 μM besides sc12, which was 10 μM. The concentration of Λ- and Δ-8 was 10 μM. Oligonucleotides and metallohelices were mixed in 1 × TBE buffer and 10 mM NaCl, left for 15 min at RT, and then analyzed on a 15% PAA native gel run at 6°C. 3WJ, dup, ss, and hp correspond to assembled 3WJ, duplex, single strand and hairpin, respectively,

Figure 5.

Fluorescence titrations of Λ- and Δ-8 (1 μM) with increasing concentrations (indicated in the figure) of DNA 3WJ (upper panels) and duplex (lower panels). Buffer conditions were 10 mM sodium phosphate buffer (pH 7), 100 mM NaCl, and 5 mM MgCl₂.

Figure 6.

(A) Scheme of the formation of 1D architecture via association of 3WJs stabilized by Λ-8. (B, C) Representative AFM images of 3WJ-forming oligonucleotides (1 × 10⁻⁷ M, each) in the absence (B) and presence (C) of 1 × 10⁻⁷ M Λ-8.

Figure 7.

Representative confocal microscopic images of H2AX phosphorylation in HCT116 cells non-treated (control) or treated with 1 × IC₅₀ of Λ-8 or Δ-8 for 24 h. The cells were fixed and stained with primary anti-γH2AX antibody and Alexa Fluor 488 conjugated secondary antibody. Cell nuclei were counterstained with DAPI.

Figure 8.

Number of γH2AX foci per cell after treatment of HCT116 cells with 1 × IC₅₀ of Λ- and Δ-enantiomers of 8 and 6 for 24 h. γH2AX foci were manually scored in at least 100 cell nuclei in 20 images. Each dot represents the average number of foci per cell in one image.