Selective recognition of G-quadruplex telomeric DNA by a bis(quinacridine) macrocycle

The interaction of G-quadruplex DNA with the macrocyclic compound BOQ1, which possesses two dibenzophenanthroline (quinacridine) subunits, has been investigated by a variety of methods. The oligonucleotide 5′-A(GGGT₂A)₃G₃, which mimics the human telomeric repeat sequence and forms an intramolecular quadruplex, was used as one model system. Equilibrium binding constants measured by biosensor surface plasmon resonance (SPR) methods indicate a high affinity of the macrocycle for the quadruplex conformation (K > 1 × 10⁷ M^-1) with two equivalent binding sites. The affinity of BOQ1 for DNA duplexes is at least 1 order of magnitude lower. In addition, the macrocycle is more selective than the monomeric control compound (MOQ2), which is not able to discriminate between the two DNA structures (K_duplex ≈ K_quadruplex ≈ 10⁶ M^-1). Strong binding of BOQ1 to G4 DNA sequences was confirmed by fluorometric titrations with a tetraplex-forming oligonucleotide. Competition dialysis experiments with a panel of different DNA structures, from single strands to quadruplexes, clearly established the quadruplex binding specificity of BOQ1. Fluorescence resonance energy transfer (FRET) T_m experiments with a doubly labeled oligonucleotide also revealed a strong stabilization of the G4 conformation in the presence of BOQ1 (ΔT_m = +28 °C). This ΔT_m value is one of the highest values measured for a G-quadruplex ligand and is significantly higher than observed for the monomer control compounds (ΔT_m = +10-12 °C). Gel mobility shift assays indicated that the macrocycle efficiently induces the formation of G-tetraplexes. Strong inhibition of telomerase was observed in the submicromolar range (IC₅₀ = 0.13 μM). These results indicate that macrocycles represent an exciting new development opportunity for targeting DNA quadruplexes.