PNA-nitrogen mustard conjugates are effective suppressors of HER-2/neu and biological tools for recognition of PNA/DNA interactions.
Peptide nucleic acids (PNAs) are promising tools for gene regulation. One of the challenges of using PNAs as gene regulators is the need to optimize the efficiency of interaction with critical sequences of DNA. To improve the efficiency of binding between PNAs and the HER-2/neu promoter, mono- and bis-pyrimidine-rich PNAs were conjugated to a nitrogen mustard at either the amino or carboxy terminus. Gel shift analysis demonstrated that conjugation to an alkylating agent slowed PNA binding and favored PNA:DNA:DNA triplex helix formation while preserving a high binding affinity. Sites of DNA alkylation were visualized by piperidine cleavage and showed PNA binding first by Hoogsteen bond formation with the target duplex to form a stable PNA:DNA:DNA triplex structure which is later converted to a PNA:DNA:PNA triple helix by strand invasion and Watson-Crick base pairing by a second PNA molecule. In this way, PNA-directed DNA alkylation was used to deduce the mode of PNA binding. Transient transfection experiments demonstrated that the PNA-nitrogen mustard conjugates suppressed HER-2/neu expression by up to 80%. In comparison with an unmodified mono-PNA or a bis-PNA, these results indicate that the covalent adducts stabilized PNA binding in cells and suggest that the conjugation of PNAs to nitrogen mustards is a robust strategy for developing antigene PNA oligonucleotides to prevent transcription.
Zhilina
Zhanna V
ZV
Arizona Cancer Center, University of Arizona, 1515 North Campbell, 85724, Tucson, Arizona, USA.
Ziemba
Amy J
AJ
Nielsen
Peter E
PE
Ebbinghaus
Scot W
SW
D018906:Antineoplastic Agents, Alkylating / Q000737:chemistry / Q000494:pharmacology; D002699:Chlorambucil / Q000737:chemistry / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry; D015536:Down-Regulation; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D009588:Nitrogen Mustard Compounds / Q000737:chemistry / Q000494:pharmacology*; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018906:Antineoplastic Agents, Alkylating; D002699:Chlorambucil; D004247:DNA; D009588:Nitrogen Mustard Compounds; D020135:Peptide Nucleic Acids; D018719:Erb-b2 Receptor Tyrosine Kinases; C008826:phenacid