We report sedimentation velocity and equilibrium measurements performed with an analytical ultracentrifuge to elucidate the effects of limited flexibility on the transport properties of semiflexible, monodisperse, double-stranded, blunt-ended DNA restriction fragments. We study a homologous series of fragments with 400, 800, and 1600 base pairs (3 to 11 persistence lengths), which are specifically designed and synthesized for this purpose (Part 1). The molecular weights following from the sedimentation measurements agree well with the values expected on the basis of the number of base pairs. The sedimentation coefficients at infinite dilution are in good agreement with theoretical predictions for wormlike cylinders. The first order in volume fraction (phi) coefficient K of the phi-dependent sedimentation coefficient s(phi) = 1 - K phi decreases from 1178 for the shortest fragment to 882 for the longest fragment. These values are much larger than predicted for uncharged rigid rods, indicating the presence of associates with an enhanced aspect ratio and excluded volume. The precise match of the molecular weights obtained from exponential sedimentation-diffusion equilibrium distributions with weights calculated from the number of base pairs shows that any association is reversible and disappears at sufficiently low DNA concentration. (c) 2005 Elsevier Inc. All rights reserved.