Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR

Abstract

We have performed solid-state 31P-19F REDOR nuclear magnetic resonance (NMR) experiments to monitor changes in minor groove width of the oligonucleotide d(CGCAAA2'FUTGGC)*d(GCCAAT(pS)TT GCG) (A3T2) upon binding of the drug distamycin A at different stoichiometries. In the hydrated solid-state sample, the minor groove width for the unbound DNA, measured as the 2'FU7-pS19 inter-label distance, was 9.4 +/- 0.7 A, comparable to that found for similar A:T-rich DNAs. Binding of a single drug molecule is observed to cause a 2.4 A decrease in groove width. Subsequent addition of a second drug molecule results in a larger conformational change, expanding this minor groove width to 13.6 A, consistent with the results of a previous solution NMR study of the 2:1 complex. These 31P-19F REDOR results demonstrate the ability of solid-state NMR to measure distances of 7-14 A in DNA-drug complexes and provide the first example of a direct spectroscopic measurement of minor groove width in nucleic acids.

Figure 1

Chemical structure of the minor groove binder distamycin.

Figure 2

(A) The A₃T₂ DNA sequence used in this study. ^2′FU indicates 2′-fluoro-2′-deoxyuridine and the dot between T18 and T19 denotes a phosphorothioate. (B) Expanded view of A₃T₂ DNA showing label positions corresponding to standard and P-F REDOR measures of minor groove width across the central A₃T₂ tract. The dark sphere at upper right marks the i phosphate (T19); on the lower left, the dark sphere indicates the i + 3 phosphate (T8), while the light sphere indicates the 2′F label used in the present experiments. The standard B-form DNA model for the A₃T₂ oligomer was generated using the biopolymer module of Insight II (Biosym). (C) Stereoview of the free A₃T₂ DNA generated using Insight II. Dark spheres show location of phosphorothioate and ^2′FU.

Figure 3

REDOR dephasing curves for A₃T₂ DNA and its 1:1 and 2:1 distamycin complexes. Solid lines represent expected decay curves based on simulations. Diamonds mark data for the unbound A₃T₂ DNA, triangles for the 1:1 distamycin:A₃T₂ complex and the square for the 2:1 distamycin:A₃T₂ complex. The lowest (7.0 Å) line represents a simulation for the 1:1 sample, where the fit is a weighted superposition of simulated dephasing curves corresponding to the three species present in the sample (unbound A₃T₂ and the 1:1 and 2:1 complexes), as described in the text.

Figure 4

REDOR S and S_o spectra for the 2:1 distamycin:A₃T₂ DNA complex. Plots were generated without line broadening and show absolute intensities. (A) Reference (S_o) ³¹P spectrum, where pS denotes the phosphorothioate signal. The large central peak and its spinning side bands (SSB) are due to unmodified backbone phosphates. (B) Experimental (S) REDOR spectrum, showing diminished ³¹P backbone and phosphorothioate peak intensities following application of ¹⁹F pulses.