Interaction of Artificial Nuclease and DNA: Atomic Force Microscopy Data

Abstract

The interest in studies of nucleic acid cleavage under the action of nucleases is largely related to the development of new systems for solving problems of genetic engineering and to the search for new-generation therapeutic drugs.In this work, the highly efficient cleavage of plasmid DNA by original synthetic biodegradable and biocompatible nucleases, conjugates of natural products, was studied by different physicochemical methods. The invoking of atomic force microscopy (AFM) as an additional method allowed us to improve visualization and find reliable evidence for full nonspecific cleavage of the nucleic acid under the action of the artificial nucleases and to interpret more correctly the data of gel electrophoresis and electronic spectroscopy.The destruction of nucleic acids can proceed enzymatically, i.e., under the action of the extensive group of nucleases of natural origin. A substantial number of artificial nucleases, which are compounds of various classes or conjugates, are also known [1, 2].The search for and design of biocompatible and biodegradable compounds able to perform highly efficient destruction of DNA and RNA are currently under way. Among the known agents, mention should be made of the hemin conjugates with peptides (hemin peptides) we synthesized previously [3], in particular, those described by the formula N N N N Fe 3+ COOH COX Cl -(OH) (X) (I) X = -ArgArgTrpHisArgLeuLysGlu(OMe)OH (II) X = -ArgTrpHisArgLeuLysGlu(OMe)OH (III) X = -TrpHisArgLeuLysGlu(OMe)OH The peptide components of hemin peptides are fragments of the HL-60 cell line of human promyelocyte leukemia differentiating factor (HLDF) [2].The data on the nuclease activity of compounds I -III were obtained by the traditional method, namely, gel electrophoresis in agarose gel with coloring of various forms of the plasmid DNA pGem by a fluorescent intercalating dye, ethidium bromide [3, 4] (Fig. 1). The plasmid DNA pGem is a two-strand ring molecule (3700 base pairs).Hemin peptides I -III are able to cleave all forms of plasmid DNA, the cleavage efficiency and conditions (hemin peptide concentration, pH, and medium composition) depending on their structure.