Inhibition of Neomycin Phosphorotransferase Expression in Entamoeba histolytica with Antisense Peptide Nucleic Acid (PNA) Oligomers

Abstract

PNA are DNA analogs in which the entire phosphate-sugar backbone has been replaced by an isomorphic pseudopeptidic chain to which the four bases are linked. They have been shown to bind complementary natural polynucleotide sequences with higher affinity than their natural counterparts, partially due to the high flexibility and absence of charge of the artificial backbone. Furthermore, their artificial structure is not recognized by proteases and nucleases, making PNA exceptionally stable in biological environments (1). This consideration is of particular relevance to Entamoeba histolytica , as it possesses numerous hydrolases, some membrane bound. PNA oligomers have been used in the last few years as antisense and antigene agents in a variety of cell-free and cellular systems, showing that they can effectively and selectively downregulate gene expression at the translational or transcriptional levels when used at micromolar concentrations (2). The difficulties inherent in the cultivation of E. histolytica trophozoites and the lack of procedures for deletion of genes have made it very difficult to perform classical genetic studies aimed at the characterization of phenotypes resulting from gene inactivation. Only recently, antisense strategies using plasmids transcribing antisense mRNAs have been successfully used, allowing the downregulation of specific genes and the analysis of resulting phenotypes (3). In this study, we report the use of PNA oligomers as antisense agents for effective and specific downregulation of expression of the bacterial gene neomycin phosphorotransferase (NPT) in E. histolytica in culture, with results comparable to antisense strategies recently reported. In contrast to other cellular types tested, notably mammalian cells, E. histolytica is permeable to unmodified PNA, and inhibition of gene expression is significant and easily achieved in a dose-dependent manner, saving the work of cloning, transfection, and selection procedures needed for plasmid-mediated antisense inhibition.