1H nuclear magnetic resonance study of the dynamic properties of the B and Z-forms of poly[d(A-br 5C) · d(G-T)

Abstract

Poly[d(A-br 5C) · d(G-T)], a synthetic polynucleotide with a 50% A · T base composition, undergoes a reversible, highly co-operative transition between the right-handed B and left-handed Z conformations. The latter is stabilized at both elevated temperature and ionic strength. The B and Z-forms of poly[d(A-br 5C) · d(G-T)] coexist in 4.6 m-NaCl at 45 °C. Due to slow exchange, two sets of T im and G im resonances are observed and can be assigned to the B and Z conformations (the chemical shifts are, respectively, T im = 13.4, 14.1 p.p.m. (parts/million); and G im = 11.9, 12.4 p.p.m.). Measurements of the 1H spin-lattice ( R 1) and spin-spin ( R 2) relaxation rates of the exchangeable thymine (T im) and guanine (G im) imino protons have been used to probe the internal dynamics of the B and Z-forms of poly[d(A-br 5C) · d(G-T)] and the mechanism of the B- Z transition. The proton exchange behavior in the B and Z conformations is quite different. At elevated temperature, R 1 for both T im and G im in the B conformation is dominated by exchange with the solvent, with T im exchanging more rapidly than G im. This demonstrates that exchange involves the opening of single base-pairs and that neighboring A · T and G · br 5C base-pairs exchange independently of each other. B-form poly[d(A-br 5C) · d(G-T)] is unusual in that there is an acceleration of the T im exchange rate with increasing NaCl concentration. Conversion to the Z-form by addition of 4.5 m-NaCl dramatically reduces both the T im and G im exchange rates (estimated to be less than 2 s −1 at 70 °C). Thus, the G · br 5C base-pair and, in particular, the A · T base-pair are stabilized in the Z conformation. By measuring relaxation rates at 45 to 50 °C where the B and Z-forms are in equilibrium, we find that the B- Z interconversion rates are less than two per second. In the B conformation at 25 °C, the dipolar contributions to the imino proton relaxation rates are about one-third of those expected on the basis of a rigid rod model for 65 base-pair fragments, a difference we assign to large amplitude (30 ° high frequency (< 100 ns) out-of-plane motions of the bases. Conversion to the Z conformation has little effect on the dipolar contributions to relaxation, i.e. on the internal motions. In both the B and Z-forms at 25 °C the measured imino proton linewidths are about twice those calculated from R 2, possibly due to multiple conformational states that are in slow exchange on the n.m.r. †† †† Abbreviations used: n.m.r., nuclear magnetic resonance; DSS, sodium 4,4-dimethyl-4-silapentane-1-sulfonate; p.p.m., parts per million. time scale.