Nuclear magnetic resonance and optical experiments are combined to determine the rms amplitude of local angular motion of purines in DNA in solution. A 12 base-pair duplex DNA with the sequence d(CGCGAATTCGCG)2 is deuterated at the H8 positions of adenine and guanine by exchange with solvent at 55 degrees C. The deuterium nmr spectrum of this DNA is measured at 30 mg/mL at 30 degrees C in an 11.76 Tesla magnet (76.75 MHz). The time-resolved fluorescence polarization anisotropies (FPA) of this same sample and also a greatly diluted sample (0.215 mg/mL) were measured after addition of ethidium. FPA measurements of the dilute sample yield the hydrodynamic radius, RH = 9.94 +/- 0.2 A, while those at the nmr concentration are employed to characterize the collective motions in terms of either an enhanced viscosity or dimer formation. The rms amplitude of local angular motion was determined by analyzing the 2H-nmr spectrum, in particular the line width, using recently developed theory for the transverse relaxation rate (RQ2) together with essential information about the collective motions from these and other optical studies. When the principal-axis frame of the electric field gradient tensor is assumed to undergo overdamped libration around each of its three body-fixed axes in an isotropic deflection potential, then the rms amplitude of local angular motion around any single axis is found to lie in the range 10 degrees-11 degrees, provided the high DNA concentration acts to enhance the viscosity, and is about 9 degrees-11 degrees, if it acts to produce end-to-end dimers. The proton nmr relaxation data of Eimer et al. are reanalyzed and shown to yield an rms amplitude of angular motion of the cytosine H5-H6 internuclear vector of 9 degrees-10 degrees, depending upon its orientation with respect to the helix axis. In all of these analyses, full account is taken of the collective twisting and bending deformations, which have a small but significant effect on the results. It is shown that the rms amplitudes of local angular motion do not depend strongly on the model (potential), provided that isotropic rotation around the same number of axes is allowed and that one compares rms angles of the same dimensionality. The rms amplitudes of local angular motion in solution are comparable to those observed for the same sequence at low levels of hydration in the solid state.

SynopsisOptical anisotropy data spanning a very wide time range are analyzed using a recently developed theory for filamentous macromolecules that can bend, twist, and also admit overdamped local libration (or wobble) of the chromophore. A rapid relaxation in the fluorescence polarization anisotropy (FPA) near s is fitted well by superimposing isotropic wobble of the chromophore (7" rms polar and azimuthal amplitude) on the long-wavelength twisting and bending motions that characterize the relaxation at longer times but not by the latter alone. Moreover, the decay of the FPA from 0.5 to 150 ns cannot be satisfactorily fitted by chromophore wobble in an otherwise rigid DNA and must be assigned primarily to twisting, as noted previously.Data from 26 ns to 20 ps for 600 base-pair DNA are accurately fitted with only a single adjustable scaling factor when the tumbling correlation function is taken to be the empirical electric birefringence decay function of Elias and Eden. The BarkleyZimm (BZ) tumbling correlation for very long filaments appears to decay too rapidly and results in significant overestimation of the depolarization for t 5 300 ns. In the range of the FPA experiments ( t 2 150 ns), equally good fits with equally uniform torsion constants are obtained for long DNAs, whether one assumes the BZ tumbling correlation function or neglects tumbling entirely, but the best-fit torsion constant (actually the product of the torsion constant and friction factor) is increased by the factor 1.9 when the BZ result is used with a persistence length of a = 500 A. The BZ bending theory is compared with other experimental data, and also with a simulation at very short times with mixed results. Present uncertainties regarding the tumbling dynamics and the friction factor for azimuthal rotation allow the torsion constant to be as much as 3.8 times larger than the initial estimate of Thomas et al. Apparent torsion constants obtained from relative ligase kinetics measurements are also briefly discussed.