Time-resolved fluorescence polarization anisotropy of short restriction fragments: the friction factor for rotation of DNA about its symmetry axis.

Abstract

AbstractThe time‐resolved fluorescence polarization anisotropy (FPA) of ethidium dye intercalated in 43 and 69 base pair (bp) restriction fragments is measured, and the friction factor per bp for rotation of DNA about its symmetry axis is determined. The same value of the hydrodynamic radius, a = 12.0 ± 0.6 Å, is obtained for both the 43‐ and 69‐bp fragments, but only when (1) the twisting correlation functions appropriate for such short filaments are used: (2) the correct amplitude is employed for the uniform tumbling mode decays: and (3) the data analysis is restricted to times after the internal bending modes have died away leaving just reduced amplitudes of the exponentially decaying uniform tumbling modes. The present value of the hydrodynamic radius is significantly larger than that implied by the cross‐sectional area perpendicular to the symmetry axis. This strongly suggests that a significant fraction of water in the major and minor groves is rotating more or less rigidly with the DNA on this time scale. The correct expression for the amplitude Dn(∞) of the uniform mode decay of the tumbling correlation function, including the average over all sites to which the dye could bind, is derived in the appendix. The present theory for Dn(∞) is compared with that of Barkley and Zimm [(1979) J. Chem. Phys. 70, 2991–3007], and with recent Brownian simulations of discrete wormlike chains by Allison and co‐workers [S. A. Allison and J. A. McCammon (1984) Biopolymers 23, 363–375; S. A. Allison (1986) Macromolecules 19, 118–124].