Direct NMR Detection of Alkali Metal Ions Bound to G-Quadruplex DNA

Abstract

We describe a general multinuclear (¹H, ²³Na, ⁸⁷Rb) NMR approach for direct detection of alkali metal ions bound to G-quadruplex DNA. This study is motivated by our recent discovery that alkali metal ions (Na⁺, K⁺, Rb⁺) tightly bound to G-quadruplex DNA are actually “NMR visible” in solution (Wong, A.; Ida, R.; Wu, G. Biochem. Biophys. Res. Commun. 2005, 337, 363). Here solution and solid-state NMR methods are developed for studying ion binding to the classic G-quadruplex structures formed by three DNA oligomers: d(TG₄T), d(G₄T₃G₄), and d(G₄T₄G₄). The present study yields the following major findings. (1) Alkali metal ions tightly bound to G-quadruplex DNA can be directly observed by NMR in solution. (2) Competitive ion binding to the G-quadruplex channel site can be directly monitored by simultaneous NMR detection of the two competing ions. (3) Na⁺ ions are found to locate in the diagonal T₄ loop region of the G-quadruplex formed by two strands of d(G₄T₄G₄). This is the first time that direct NMR evidence has been found for alkali metal ion binding to the diagonal T₄ loop in solution. We propose that the loop Na⁺ ion is located above the terminal G-quartet, coordinating to four guanine O6 atoms from the terminal G-quartet and one O2 atom from a loop thymine base and one water molecule. This Na⁺ ion coordination is supported by quantum chemical calculations on ²³Na chemical shifts. Variable-temperature ²³Na NMR results have revealed that the channel and loop Na⁺ ions in d(G₄T₄G₄) exhibit very different ion mobilities. The loop Na⁺ ions have a residence lifetime of 220 μs at 15 °C, whereas the residence lifetime of Na⁺ ions residing inside the G-quadruplex channel is 2 orders of magnitude longer. (4) We have found direct ²³Na NMR evidence that mixed K⁺ and Na⁺ ions occupy the d(G₄T₄G₄) G-quadruplex channel when both Na⁺ and K⁺ ions are present in solution. (5) The high spectral resolution observed in this study is unprecedented in solution ²³Na NMR studies of biological macromolecules. Our results strongly suggest that multinuclear NMR is a viable technique for studying ion binding to G-quadruplex DNA.