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. 2003 Sep 1;31(17):5084-9.
doi: 10.1093/nar/gkg720.

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

Greg L Olsen  1 , Elizabeth A Louie, Gary P Drobny, Snorri Th Sigurdsson
Affiliations

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

Greg L Olsen et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Chemical structure of the minor groove binder distamycin.
Figure 2
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
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
Figure 4
REDOR S and So spectra for the 2:1 distamycin:A3T2 DNA complex. Plots were generated without line broadening and show absolute intensities. (A) Reference (So) 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.
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