125] Monolayer techniques in electron microscopy of nucleic acid molecules

Abstract

This chapter discusses the monolayer techniques in electron microscopy of nucleic acid molecules and describes a method in which DNA or RNA of a bulk solution is transformed to a monomolecular layer. To do this, a film of protein floated onto an aqueous solution is used. Many globular proteins in solution, and similar polymers, are capable of producing surface films on an aqueous solution in a fiat trough. As the protein is surface-denatured, it forms an insoluble film. This protein film can be considered as a monomolecular layer and assumed to exist as a molecular net of unfolded polypeptide chains. The DNA or RNA is adsorbed to it by basic side groups of amino acid residues. This adsorption effectively brings a nucleic acid molecule from a three-dimensional position in an aqueous solution (the subphase) to a two-dimensional position—that is, adsorbed to the polypeptide net. After adsorption, the monolayer, consisting of the protein net and the adsorbed nucleic acid, is itself adsorbed to a solid support and then dried. The preparation can be contrasted for electron microscopy, and electron micrographs are taken. The three procedures are employed and discussed in the chapter—a spreading procedure, a diffusion procedure, and a one-step release procedure. These three procedures explains that most globular proteins have sufficient spreading activity and these proteins become surface-denatured on top of the subphase; more information is needed as to whether DNA or RNA is enveloped as a complex of protein and nucleic acid, or more likely, is nakedly adsorbed to polypeptide chains by a few adsorption side groups per unit length; and the final concentration of nucleic acids adsorbed in the diffusion procedure depends on concentration in the subphase, period of adsorption, salt concentrations, and size of the nucleic acid.