Preparation of Double/Single-Stranded DNA and RNA Molecules for Electron Microscopy

Electron microscopy (EM) has proved to be an increasingly powerful tool in the study of nucleic acids. It provides qualitative and quantitative data on the size and structure of native and experimentally manipulated DNA and RNA molecules. The electron microscope, as a tool for analyzing molecular structure, interactions, and processes, is one that can be used with increasing confidence, particularly in conjunction with biochemical and biophysical studies (1 -4 ). In order to prepare nucleic acids for electron microscopy, there are two important requirements. Since nucleic acids are thread-like molecules which form three-dimensional flexible random coils in aqueous solution, they must converted to two-dimensional unaggregated molecules. These requirements are met by spreading them together with a surface-active substance which traps the nucleic acid molecules in a monolayer floating on a hypophase of water (1 -8 ). This mono-layer then is absorbed to a thin carbon film on a carbon-coated grid. To enhance contrast, the nucleic acid molecule is stained either with uranyl acetate (in particular, for dark-field microscopy) or by low-angle shadowing with evaporated heavy metal, for example, Pt/Pd alloy (see Fig. 1 ).