Precise Distance Measurements in DNA G‐Quadruplex Dimers and Sandwich Complexes by Pulsed Dipolar EPR Spectroscopy

DNA G‐quadruplexes show a pronounced tendency to form higher‐order structures, such as π‐stacked dimers and aggregates with aromatic binding partners. Reliable methods for determining the structure of these non‐covalent adducts are scarce. Here, we use artificial square‐planar Cu(pyridine)(4) complexes, covalently incorporated into tetramolecular G‐quadruplexes, as rigid spin labels for detecting dimeric structures and measuring intermolecular Cu(2+)–Cu(2+) distances via pulsed dipolar EPR spectroscopy. A series of G‐quadruplex dimers of different spatial dimensions, formed in tail‐to‐tail or head‐to‐head stacking mode, were unambiguously distinguished. Measured distances are in full agreement with results of molecular dynamics simulations. Furthermore, intercalation of two well‐known G‐quadruplex binders, PIPER and telomestatin, into G‐quadruplex dimers resulting in sandwich complexes was investigated, and previously unknown binding modes were discovered. Additionally, we present evidence that free G‐tetrads also intercalate into dimers. Our transition metal labeling approach, combined with pulsed EPR spectroscopy, opens new possibilities for examining structures of non‐covalent DNA aggregates.