Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

Abstract

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms.

A non-covalent peptide-based carrier for in vivo delivery of DNA mimics

“…In the present study, we have elaborated a strategy combining a non-covalent peptide-based delivery system and a charged PNA-like DNA mimic that is likely to have potent implications i n therapeutics. HypNA- p PNA DNA mimics have already been used successfully both in vitro and in vivo (19,20,31,32) and offer several advantages due to their tight interaction with target DNA as well as their high solubility (21,32). Cell-penetrating peptides are most promising tools for delivery of therapeutic molecules (8,9) and the recent reconsideration of their cellular uptake mechanism has provided new perspectives for their in vivo application (25,26).…”

Peptide Nucleic Acids with a Structurally Biased Backbone: Effects of Conformational Constraints and Stereochemistry

“…(7) [107]. These compounds have been proved to be able to perform efficient gene silencing in vitro and in vivo [108]. It is interesting that the 3'-equivalent and 5' equivalent of these compounds match with the model proposed in Fig.…”

“…In fact the best performing trans-L-isomers were chosen for the biological tests. Cell-free experiments showed the ability of these compounds to inhibit translation with antisense mechanism (as tested by inhibiting luciferase activity in cellular extracts) [108]. These compounds were found to be able to penetrate into HS-68 and HeLa cells with cytoplasmatic distribution [37,108].…”

“…Cell-free experiments showed the ability of these compounds to inhibit translation with antisense mechanism (as tested by inhibiting luciferase activity in cellular extracts) [108]. These compounds were found to be able to penetrate into HS-68 and HeLa cells with cytoplasmatic distribution [37,108]. Furthermore, they were shown to be effectively delivered into eukaryotic cells using lipofectamine (LFA) transfection, with maximum uptake after 20-24 h [108].…”

“…These compounds were found to be able to penetrate into HS-68 and HeLa cells with cytoplasmatic distribution [37,108]. Furthermore, they were shown to be effectively delivered into eukaryotic cells using lipofectamine (LFA) transfection, with maximum uptake after 20-24 h [108]. This approach is possible with these compounds since they are negatively charged and can interact with the cationic carrier.…”