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 demonstrate that artificial square-planar Cu(pyridine) 4 complexes, covalently incorporated into tetramolecular G-quadruplexes, can be used as rigid spin labels for detecting dimeric structures by 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 with the new method, and previously unknown binding modes were discovered. Additionally, we give the first distance-based evidence that free G-tetrads composed of four guanines 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.