Competitive binding of triplex-forming oligonucleotides in the two alternate promoters of the PMP22 gene.
Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.
Hai
M
M
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
Bidichandani
S I
SI
Hogan
M E
ME
Patel
P I
PI
D000818:Animals; D020029:Base Pairing; D001667:Binding, Competitive; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics / Q000378:metabolism / Q000628:therapy; D006224:Cricetinae; D004247:DNA / Q000737:chemistry; D018983:DNA Footprinting; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts / Q000378:metabolism; D015316:Genetic Therapy; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D009185:Myelin Proteins / Q000235:genetics*; D009186:Myelin Sheath / Q000502:physiology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
D009185:Myelin Proteins; D009838:Oligodeoxyribonucleotides; C073687:PMP22 protein, human; C051802:triplex DNA; D008345:Manganese; D004247:DNA