Evaluation of Transfection Protocols for Unmodified and Modified Peptide Nucleic Acid (PNA) Oligomers

Abstract

We have compared the efficacy of different transfection protocols reported for peptide nucleic acid (PNA) oligomers. A precise evaluation of uptake efficacy was achieved by using a positive readout assay based on the ability of a PNA oligomer to correct aberrant splicing of a recombinant luciferase gene. The study comprised transfection of PNA conjugated to acridine, adamantyl, decanoic acid, and porphyrine (acr-PNA, ada-PNA, deca-PNA, and por-RNA, respectively) and unmodified PNA partially hybridized to a DNA oligomer (PNA/DNA cotransfection). Furthermore, the effect of conjugation to a nuclear localization signal (NLS) was evaluated as part of the PNA/DNA cotransfection protocol. Transfection of the tested PNAs was systematically optimized. PNA/DNA cotransfection was found to produce the highest luciferase activity, but only after careful selection of the DNA oligonucleotide. Both a cationic lipid, Lipofectamine, and a nonliposomal cationic polymer, polyethylenimine (PEI, ExGen 500), were efficient transfection reagents for the PNA/DNA complex. However, Lipofectamine, in contrast to PEI, showed severe side effects, such as cytotoxicity. acr-PNA, ada-PNA, and por-PNA were transfectable with efficacies between 5 and 10 times lower than that seen with PNA/DNA cotransfection. Conjugation of PNA to NLS had no effect on PNA/DNA cotransfection efficacy. An important lesson from the study was the finding that because of uncontrollable biologic variations, even optimal transfection conditions differed to a certain extend from experiment to experiment in an unpredictable way.

Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells

“…The major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells (35). In order to solve this drawback, several approaches have been considered, including the delivery of PNA analogues with liposomes and microspheres (25,36,37).…”

Modulation of the Biological Activity of microRNA‐210 with Peptide Nucleic Acids (PNAs)

“…[30][31][32] The main limitation to the use of PNAs for altering gene expression is their low uptake by eukaryotic cells. [33] To overcome…”

Specific uptake and interactions of peptide nucleic acid derivatives with biomimetic membranes

“…6,11,15,17,[21][22] have shown promising improvements in cellular bioavailability, there is still a need to improve our understanding of cellular uptake for different 50 delivery vectors and to provide better correlations of their in vivo structure-activity relationships.…”

“…These include co-delivery with partial cDNA oligonucleotides, cationic lipids and lipophilic triphenylphosphonium (TPP) cations, and a few examples of chemical modifications of PNA oligomers with metal complexes. 6,8,[11][12][13][14][15] However, to date, the most promising 30 approach has been the conjugation of PNAs with cell penetrating peptides (CPP). 6,8,12 Although the mechanism of translocation of CPPs is not fully understood, their ability to successfully transport cargo (e.g., PNAs) into the cell makes them effective delivery agents.…”