Stereoscopic Visualization of Spermidine-DNA Torus Structure Prepared by the Freeze-Fracture, Deep-Etch Technique

Freeze-fracture TEM has been used to visualize spermidine-DNA complexes╌a model system for bacteriophage DNA packaging. We have observed the toroidal shape and bacteriophage size (770Å) of spermidine-condensed DNA particles prepared by quick freezing of DNA-spermidine solutions followed by a freeze-fracture, deep-etching technique. Single direction shadowing with low Pt-C metal levels (9Å thick) has allowed us to demonstrate that the freeze-fracture technique is capable of revealing the organization of separate double helical DNA strands on the torus surface. The spermidine-DNA toruses exhibited surface Pt-C decoration consistent with the DNA double helix being circumferentially wrapped to form the torus. In addition, we have demonstrated that a hydrated sample shows the same toroidal shapes that have been previously demonstrated by Chattoraj et. al, in a dehydrated preparation. We performed freeze-fracture TEM studies of spermidine condensed (0.2 mM) Calf Thymus DNA (5 μg/ml) complexes in 1 mM NaCl, 10 mM Tris pH 7.0. Samples were freeze-fractured and etched for 13 min at -97°C.