Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers

Abstract

Peptide nucleic acids (PNAs) may be a potent tool for gene function studies in medically important parasitic organisms, especially those that have not before been accessible to molecular genetic knockout approaches. One such organism is Entamoeba histolytica, the causative agent of amebiasis, which infects about 500 million people and is the cause of clinical disease in over 40 million each year, mainly in the tropical and subtropical world. We used PNA antisense oligomers to inhibit expression of an episomally expressed gene (neomycin phosphorotransferase, NPT) and a chromosomal gene (EhErd2, a homolog of Erd2, a marker of the Golgi system in eukaryotic cells) in axenically cultured trophozoites of E. histolytica. Measurement of NPT enzyme activity and EhErd2 protein levels, as well as measurement of cellular proliferation, revealed specific decreases in expression of the target genes, and concomitant inhibition of cell growth, in trophozoites treated with micromolar concentrations of unmodified antisense PNA oligomers.

Antigenic Epitopes Fused to Cationic Peptide Bound to Oligonucleotides Facilitate Toll-Like Receptor 9-Dependent, but CD4+ T Cell Help-Independent, Priming of CD8+ T Cells

“…Peptides with a length of six to nine R residues translocate optimally through membranes, and peptides containing R residues translocate more efficiently than peptides containing K residues. These cationic peptides efficiently deliver large, 20-to 200-kDa proteins (2,4,6), liposomes, radioisotopes, or antisense oligonucleotides (7)(8)(9)(10)(11) into cells. They thus represent a novel, potentially universal delivery system for proteins or nucleotides into the cytosol in bioactive form.…”

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

“…10 Recent work has demonstrated that cellular uptake of free PNAs can occur, although this requires very high concentrations of PNAs, except in neuronal cells, 11,12 and yields limited biological response. [13][14][15] In order to improve delivery of PNAs into cells and to reduce the doses required for detectable effects, several chemical modifications based on covalently linked cell-penetrating peptides have been successfully used. 10,12,[16][17][18] Linkage of nuclear localization or polylysine sequences to PNAs have also been reported to improve both their cellular internalization and nuclear translocation.…”

Preparation and Evaluation of 99mTc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors

“…PNA has been reported to resist nuclease and protease degradation (29) and can enhance acid specificity and inhibit mRNA expression (30,31). Furthermore, these modifications can increase the flexibility and binding affinities of PNA for its complementary single-stranded PNA, exceeding that of comparable DNAs (28).…”