Structure of a Quinobenzoxazine-G-Quadruplex Complex by REDOR NMR

Biochemistry, 2004, 43 (38), pp 11953--11958

ABSTRACT
Rotational-echo double resonance solid-state 31P{19F} and 13C{19F} NMR spectra have been used to locate the binding of a fluoroquinobenzoxazine to a DNA G-quadruplex labeled by phosphorothioation and [methyl-13C]thymidine.

(Top) Sequence and numbering of the oligomer used in this study. (Bottom) Structure of QQ58.


31P{19F} REDOR NMR spectra (202 MHz) of the complex of QQ58 with a sulfur-tagged quadruplex. The top two spectra show sidebands resulting from magic-angle spinning at 7463 Hz after 64 rotor cycles of dipolar evolution. The full-echo spectrum (no dephasing) is labeled S0 and the REDOR difference spectrum (the difference between echo spectra with and without dephasing), DS. The major set of peaks in each spectrum is due to PO4 and the minor set, to PO3S. Digitizing of the NMR signal was done at 20 times 7463 Hz so that the Fourier transform of every 20th time-domain data point resulted in the bottom two spectra. These spectra have center- and sidebands folded together in a window whose width is the spinning frequency. The major peak on the left is due to 31PO4, and the minor peak on the right is due to 31PO3S. The relative positions of the two peaks were determined by the choice of the radio frequency carrier offset. The true centerband frequency of the 31PO3S is greater (to the left in the top two spectra) than that of the 31PO4 peak by 56 ppm (11.3 kHz). Integrated intensities were measured from the Lorentzian line fits shown in red. The G-quadruplex-drug complex weighed about 8 mg and represented about 0.8 umol of binding sites. Data acquisition required 5 days of spectral accumulation.

Two views (top and side) of the NMR model (left column) and the MCT model (right column) of Duan et al. of the QQ58 quadruplex complex. The tubes represent the DNA backbones and pass through the phosphates, shown as large orange spheres. The PO3S groups are in blue (TAGGGsTTA) and red (TAGGsGTTA). The fluorine of QQ58 is shown in green, carbons in black, and the oxygens in red (all small spheres).

Orientations of the bases in the NMR and MCT models of the QQ58 quadruplex complex. G is in green,  in blue, and A in red. The thymidine methyl 13C labels are shown as isolated black spheres next to the thymidine bases. This cartoon of the complex was created in Accelrys DS Viewer Pro.