Higher-order G-quadruplex structures and porphyrin ligands: Towards a non-ambiguous relationship

Herein, we evaluated the interaction of the tetracationic porphyrin H₂TCPPSpm4 with three distinct DNA G-quadruplex (G4) models, i.e., the tetramolecular G4 d(TGGGGT)₄ (Q₁), the 5'-5' stacked G4-dimer [d(CGGAGGT)₄]₂ (Q₂), and a mixture of 5'-5' stacked G-wires [d(5'-CGGT-3'-3'-GGC-5')₄]_n (Q_n). The combined data obtained from UV-Vis, CD, fluorescence, PAGE, RLS, AFM, NMR, and HPLC-SEC experiments allowed us to shed light on the binding mode of H₂TCPPSpm4 with the three G4 models differing for the type and the number of available G4 ending faces, the length of the G4 units, and the number of stacked G4 building blocks. Specifically, we found that H₂TCPPSpm4 interacted with the shortest Q₁ as an end-stacking ligand, whereas the groove binding mode was ascertained in the case of the Q₂ and Q_n G4 models. In the case of the interaction with Q₁ and Q_n, we found that H₂TCPPSpm4 induces the formation of supramolecular aggregates at porphyrin/G4 ratios higher than 2:1, whereas no significant aggregation was observed for the interaction with Q₂ up to the 5:1 ratio. These results unambiguously demonstrated the suitability of porphyrins for the development of specific G4 ligands or G4-targeting diagnostic probes, being H₂TCPPSpm4 capable to distinguish between different G4s.