G-quadruplexes are classic DNA secondary structures formed by Hoogsteen base pairing in guanine-rich oligonucleotides resulting in stacked guanine tetrads. They have been found to regulate the expression of oncogenes and human telomeric properties in vivo and have therefore been identified as promising drug targets in anticancer research. Many small molecules and metal complexes were found to bind and stabilize the folded secondary structures thereby acting as potential anticancer drugs. DNA G-quadruplexes show a pronounced tendency to form higher-order structures such as π-stacked dimers, G-wires and aggregates with aromatic binding partners. Reliable methods for determining the structure of these non-covalent adducts are scarce. We use artificial square-planar Cu(pyridine)4 complexes, covalently incorporated into tetramolecular G-quadruplexes, as spin labels for detecting dimeric structures and measuring intermolecular Cu2+–Cu2+ distances via pulsed dipolar EPR spectroscopy. The rigidity of the incorporated Cu2+ complexes results in unprecedentedly narrow distance distributions. 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 drug-like G-quadruplex-binding ligands, 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 formed by untethered guanines or guanosines 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 and investigating DNA-protein interactions.

Precise distance measurements in DNA G-quadruplex dimers and sandwich complexes by pulsed dipolar EPR spectroscopy

ACS Spring Meeting 2021

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