Field- and phage-induced dipolar couplings in a homodimeric DNA quadruplex, relative orientation of G center dot(C-A) triad and G-tetrad motifs and direct determination of C2 symmetry axis orientation

We present a new NMR procedure for determining the three-dimensional fold o f C2-symmetric nucleic acid homodimers that relies on long-range orientatio nal constraints derived from the measurement of two independent sets of res idual dipolar couplings under two alignment conditions. The application is demonstrated on an N-15/C-13-enriched deoxyoligonucleotide sequence, d(G-G- G-T-T-C-A-G-G), shown previously to dimerize into a quadruplex in solution and form a pair of G . (C-A) triads and G-G-G-G tetrads (G-tetrad) motifs. One-bond H-1-N-15 (D-1(NH)) and H-1-C-13 (D-1(CH)) residual dipolar couplin gs have been measured between nuclei in the bases of these motifs using bac teriophage as an ordering medium, and under direct magnetic field alignment (800 MHz), By combining the two dipolar data sets in an order matrix analy sis, the orientation of the G . (C-A) tried relative to the G-tetrad within a contiguous monomeric unit can directly be determined, even in the presen ce of interstrand/intrastrand NOE ambiguity. We further demonstrate that th e orientation of the C2-axis of molecular symmetry in the homodimer relativ e to the G . (C-A) triad and G-tetrad motifs can unambiguously be determine d using the two sets of independent dipolar coupling measurements. The thre e-dimensional fold of the homodimer determined using this procedure is very regular and in excellent agreement with a previously determined high-resol ution NOE-based NMR structure, where interstrand/intrastrand NOEs were trea ted as ambiguous and where noncrystallographic symmetry constraints were im plicitly imposed during the structure calculation.