Subject(s)
Genes, ras , Oligodeoxyribonucleotides, Antisense/pharmacology , Polyethyleneimine , Transfection/methods , Blotting, Northern/methods , Flow Cytometry/methods , Fluorescent Dyes , Humans , Indicators and Reagents , Microscopy, Confocal/methods , Oligodeoxyribonucleotides, Antisense/chemical synthesis , Oligodeoxyribonucleotides, Antisense/pharmacokinetics , Phosphatidylethanolamines , RNA, Messenger/genetics , Thionucleotides , Tumor Cells, CulturedABSTRACT
We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. In order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA-RNA hybrid (Tm=86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-ras mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex with a central mismatch had little effect on mRNA translation. Codon 12 is located close to the translation initiation site and hybridization of the PNA at this position may interfere with the assembly of the translation initiation complex. To test whether polypeptide chain elongation can also be blocked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 regions. These PNAs form equally stable duplexes as that formed by the PNA targeted to the codon 12 region (ten G.C base-pairs out of 13). We show that PNA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it is possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G+C-rich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongation.
Subject(s)
Antisense Elements (Genetics)/pharmacology , Oncogene Protein p21(ras)/genetics , Peptide Chain Elongation, Translational/drug effects , Peptide Nucleic Acids/pharmacology , RNA, Messenger/genetics , Amides/chemistry , Animals , Antisense Elements (Genetics)/metabolism , Base Sequence , Binding Sites , Codon, Initiator/chemistry , Genes, ras , Humans , Kinetics , Nucleic Acid Conformation , Oncogene Protein p21(ras)/drug effects , Peptide Nucleic Acids/metabolism , Phosphoric Acids/chemistry , Point Mutation , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RabbitsABSTRACT
Phosphodiester oligodeoxyribonucleotides linked to an intercalating agent or a dodecanol tail or both complementary to the 12th codon region of Ha-ras mRNA were compared with the unmodified oligonucleotides of the same size and sequence with respect to their ability to induce RNaseH cleavage and antisense activity in cell culture. The hydrophobic tail not only protected the oligonucleotide from nucleases but also enhanced RNase H cleavage of the target. Oligonucleotides carrying both an acridine and a dodecanol substituent inhibited the proliferation of HBL100ras1 cells (human mammary cells stably transformed with the T24 Ha-ras gene carrying a G-->T point mutation in codon 12) at a 20-fold to 30-fold lower concentration than unmodified ones. Therefore, these modified oligonucleotides may prove useful for antisense applications.
