¹H‐ and ³¹P‐nmr studies of ditercalinium binding to a d(GCGC)₂ and d(CCTATAGG)₂ minihelices: A sequence specificity study

Abstract

The structures of the complexes formed in aqueous solution between ditercalinium, a bis-intercalating drug, and both the self-complementary tetranucleotide d(GCGC)2 and octanucleotide d(CCTATAGG)2, have been investigated by 400-MHz 1H-nmr and 162-MHz 31P-nmr. All the nonexchangeable protons, as well as the exchangeable imino protons and the phosphorus signals, have been assigned. Both oligonucleotides have been shown to adopt a right-handed B-DNA type structure. The addition of ditercalinium to the oligonucleotides lead to the formation of complexes in slow exchange at the nmr time scale with the free helices. At all drug-to-helix ratios studied, the ditercalinium was found in the bound form, whereas free and complexed oligonucleotides were in slow exchange, allowing resonance assignments through two-dimensional chemical exchange experiments. for d(GCGC)2 the strong upfield shifts induced on all aromatic protons of both the bases and the drug by complexation with ditercalinium suggest an interaction by intercalation of the two rings. However, the loss of twofold symmetry upon binding, as well as the chemical shift variation of the drug proton signals of one of the chromophores with temperature and concentration, favor a model in which the drug-nucleotide complexes have one ring of the drug intercalated and the other stacked on top of the external base pair. The intermolecular contacts between drug protons and nucleotide protons give a defined geometry for complexation that is consistent with the proposed model. In contrast, with d(CCTATAGG)2 several drug-nucleotide complexes were formed and a large increase in line broadening was observed at high drug-to-DNA ratios, precluding a detailed analysis of these complexes. However, the large upfield shift in the imino proton resonances together with the shielding of the ditercalinium ring protons favor a model with bis-intercalation of ditercalinium. This model is supported by the downfield shift of at least 4 out of 14 phosphorus signals. The results are compared with those obtained on ditercalinium binding to the homologous sequences d(CGCG)2 and d(TTCGCGAA)2, and discussed in terms of sequence specificity.

Tight Binding of the Antitumor Drug Ditercalinium to Quadruplex DNA

“…[22,23] This binding cavity lies in the 5'-AG sequence, a dinucleotide step frequently encountered within ditercalinium binding sites on duplex DNA [6] as well as in the octanucleotide sequence d(CCTATAGG) 2 used for NMR spectroscopy studies. [7] The 5'-AG step may perhaps be exploited by ditercalinium as its strongest binding site, with the AG base pair sandwiched between the two tetracyclic units. It is also conceivable that the ditercalinium stacks onto an external G4 tetrad, as previously observed with an external GC base pair in a short duplex d(GCGC) 2 , [7] and this may account for the second, weaker ditercalinium binding site on the G4 DNA structure.…”

“…[7] The 5'-AG step may perhaps be exploited by ditercalinium as its strongest binding site, with the AG base pair sandwiched between the two tetracyclic units. It is also conceivable that the ditercalinium stacks onto an external G4 tetrad, as previously observed with an external GC base pair in a short duplex d(GCGC) 2 , [7] and this may account for the second, weaker ditercalinium binding site on the G4 DNA structure. Specific structural studies at the atomic level, for example by high-resolution NMR spectroscopy, will be required to investigate further the geometry of the ditercalinium ± telomeresequence complex.…”

“…[7] It has also been suggested that ditercalinium can recognize and stabilize the A-type DNA conformation adopted by d(GCGCGC) 2 . [8] Therefore, it may be that ditercalinium should be considered a structure-selective rather than a sequence-selective DNA binding agent.…”

Molecular Dynamics Simulations of the Bis-Intercalated Complexes of Ditercalinium and Flexi-Di with the Hexanucleotide d(GCGCGC)2: Theoretical Analysis of the Interaction and Rationale for the Sequence Binding Specificity

“…In contrast, binding of ditercalinium to d(GCGC) 2 showed one ring of the drug intercalated at the CpG step and the other stacked on top of either of the external base pairs. 11 In both cases, however, the convex edge of the chromophores was oriented toward the minor groove. This orientation places the linker diagonally across the major groove and ensures that the chromophores adopt antiparallel orientations relative to each other, which confers the molecule an inverted-Z shape when seen from the major groove ( Figure 2).…”

“…On the other hand, binding of ditercalinium to d(CCTATAGG) 2 does not lead to a unique complex and extensive line broadening is observed, indicative of nonspecific binding despite the alternation of 5′-Pyr-Pur-3′ in the central region. 11 The preference for intercalation at 5′-CpG-3′ steps of the majority of monointercalators (with one notable exception in actinomycin D, which binds to a GpC step) 12 is supported by ample experimental evidence [13][14][15] and was not unexpected for ditercalinium. This preference is generally attributed to the lower energy required for unstacking this sequence relative to others.…”