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 i nherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A) . In an attempt to alter PMP22 gene expression as a possible therapeutic st rategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene prom oters, 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 fo r binding of these targets by TFO vs. protein(s) was compared by exposing p roteins to their target sequences after triplex formation (passive competit ion) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competiti vely interfere with the binding of protein to region I. Oligonucleotides di rected to region V competed for protein binding by a nontriplex-mediated me chanism, most likely via the formation of higher-order, manganese-destabili zable structures. Given that the activity of the P1 promoter is closely lin ked to peripheral nerve myelination, TFO identified here could serve as use ful reagents in the investigation of promoter function, the role of PMP22 i n myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter m ay have wider implications for the use of such oligonucleotides in vivo.