Targeting and regulation of the HER-2/neu oncogene promoter with bis-peptide nucleic acids.
Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.
Ziemba
Amy J
AJ
Arizona Cancer Center, Tucson, AZ 85724, USA.
Zhilina
Zhanna V
ZV
Krotova-Khan
Yulia
Y
Stankova
Lenka
L
Ebbinghaus
Scot W
SW
D000477:Alkylating Agents / Q000494:pharmacology; D001483:Base Sequence; D015139:Blotting, Southern; D002412:Cations; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008466:Mechlorethamine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D020135:Peptide Nucleic Acids / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D000477:Alkylating Agents; D002412:Cations; D004247:DNA; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D004072:Digitonin; D008466:Mechlorethamine; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D018719:Erb-b2 Receptor Tyrosine Kinases