Structural Basis for Targeting T:T Mismatch with Triaminotriazine-Acridine Conjugate Induces a U-Shaped Head-to-Head Four-Way Junction in CTG Repeat DNA

Abstract

The potent DNA-binding compound triaminotriazine-acridine conjugate (Z1) functions by targeting T:T mismatches in CTG trinucleotide repeats that are responsible for causing neurological diseases such as myotonic dystrophy type 1, but its binding mechanism remains unclear. We solved a crystal structure of Z1 in a complex with DNA containing three consecutive CTG repeats with three T:T mismatches. Crystallographic studies revealed that direct intercalation of two Z1 molecules at both ends of the CTG repeat induces thymine base flipping and DNA backbone deformation to form a four-way junction. The core of the complex unexpectedly adopts a U-shaped head-to-head topology to form a crossover of each chain at the junction site. The crossover junction is held together by two stacked G:C pairs at the central core that rotate with respect to each other in an X-shape to form two nonplanar minor-groove-aligned G·C·G·C tetrads. Two stacked G:C pairs on both sides of the center core are involved in the formation of pseudo-continuous duplex DNA. Four metal-mediated base pairs are observed between the N7 atoms of G and Co^II, an interaction that strongly preserves the central junction site. Beyond revealing a new type of ligand-induced, four-way junction, these observations enhance our understanding of the specific supramolecular chemistry of Z1 that is essential for the formation of a noncanonical DNA superstructure. The structural features described here serve as a foundation for the design of new sequence-specific ligands targeting mismatches in the repeat-associated structures.

Figure 1.

(a) Chemical structure of triaminotriazine acridine conjugate (ligand Z1). (b) Overall crystal structure of d[TT(CTG)₃AA] complexed to Z1 in an asymmetric unit. Each DNA chain is shown in cartoon representation. Chain A is colored in green, chain B in cyan, chain C in pink and chain D in yellow. (c) Schematic representation of the overall crystal structure. (d) The core of the complex formed by a crossover of all four chains in G5-C6-T7-G8-C9 penta-sequence. (e) Two central stacked G:C pairings form two GC tetrads labeled as GC-I and GC-II. The additional stacked G:C pairing formed by adjacent G8pC9 bases of chains A and C are denoted as GC-III and GC-IV, respectively. The hydrogen bonding between the bases is shown with a black dotted line.

Figure 2.

The pseudo DNA duplex of the Z1-TTT complex structure viewed from (a) front and (b) side view. A pseudo-continuous right-handed duplex DNA is formed by the complementary base pairing between A and C chains and B and D, respectively shown as cartoon representation. The Z1 intercalates through the minor groove side into the 5’-C3pT4 step shown in blue stick and surface.

Figure 3.

(a) 2F_o-F_c map for the G5-Co^II-G8 pair formed by each chain contoured at 1.0 σ. The coordinated metal ions (orange sphere) and water molecules (red sphere) appear clearly in the refined structure. Coordination and hydrogen bonds are shown by dashed lines. (b) 2F_o-F_c map for the water mediated stabilization of the central core residues. The water molecules are shown by red spheres and the hydrogen bonding is shown as dotted black lines. (c) The water W56 and W206 mediated stabilization of the central core residues. Enlarged view of W18 (d) and W19 (e) mediated hydrogen bonding between the G5 and G8 residues of each chain. The water molecules are shown by red spheres and the hydrogen bonding is shown as dotted black lines.

Figure 4.

Overall structure of Z1 binding site in the Z1-TTT complex. (a) Intercalation of Z1 through minor groove forms hydrogen bonding interactions with the T10 and G5 bases shown by black dotted lines. (b) The acridine moiety forms stacking interactions with the G11 base and the triaminoazine stacks on the G8 base. (c) 2F_o-F_c map for the three water mediated interactions between Z1 and DNA bases. The water molecules are shown by red spheres and the hydrogen bonding is shown as dotted black lines. (d) Hydrophobic interactions between Z1 and DNA bases as shown by the LIGPLOT⁺.

Figure 5.

Comparison of the DNA topologies in different sequence context associated with ligand binding in four-way junction like structures. (a) Intercalation of Z1 to T:T mismatches in CTG repeat DNA resulted in U-shaped head to head four way junctions (4WJs) like topology. (b) hydroxymethyl-trimethylpsoralen (HMT) induces a Holliday junction (HJ) formation by cross-linking the thymine bases to cause stacked-X topology. (c) Binding of platinum complex [Pt(H₂bapbpy)]-(PF₆)₂ to non-continuous pseudo duplexes causes cytosine base flipping to generate a pseudo-4WJ like crossover topology. (d) A bis(9-aminoacridine-4-carboxamide) ligand selectively recognizes the antiparallel stacked-X shaped Holliday junctions (HJ). The lower figure shows a schematic representation of each DNA topology due to different ligand binding. The respective PDB IDs for each structure are given in bold, red letters.