Evaluation of transfection protocols for unmodified and modified peptide nucleic acid (PNA) oligomers.
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.
Rasmussen
Frank Winther
FW
Pantheco A/S, DK-2970 Hørsholm, Denmark.
Bendifallah
Nadia
N
Zachar
Vladimir
V
Shiraishi
Takehiko
T
Fink
Trine
T
Ebbesen
Peter
P
Nielsen
Peter E
PE
Koppelhus
Uffe
U
D002452:Cell Count; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry* / Q000235:genetics; D014162:Transfection / Q000379:methods*
D008055:Lipids; D019913:Nuclear Localization Signals; D009838:Oligodeoxyribonucleotides; D020135:Peptide Nucleic Acids; D008156:Luciferases