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 pi-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 Cu2+-Cu2+ 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.

Automated summary

DNA G-quadruplexes have a tendency to form higher-order structures, and square-planar Cu(pyridine)(4) complexes are used as rigid spin labels to detect dimeric structures and measure intermolecular distances via pulsed dipolar EPR spectroscopy. Different spatial dimensions of G-quadruplex dimers and new binding modes of well-known G-quadruplex binders were discovered in this study. The transition metal labeling approach combined with pulsed EPR spectroscopy opens new possibilities for examining structures of non-covalent DNA aggregates.