Conditional modulation of membrane protein expression in cultured cells mediated by prion protein recognition of short phosphorothioate oligodeoxynucleotides.

We demonstrate that the levels of native as well as transfected prion protein (PrP) are lowered in various cell lines exposed to phosphorothioate oligodeoxynucleotides (PS-DNA) and can be rapidly reverted to their normal amounts by removal of PS-DNA. This transient modulation was independent of the glycosylation state of PrP, and in addition, all three PrP glycoforms were susceptible to PS-DNA treatment. Deletion of the N-terminal domain (amino acids 23-99), but not of the other domains of PrP, abrogated its PS-DNA-mediated down-regulation. PrP versions localized in the mitochondria, cytoplasm, or nucleus were not modulated by PS-DNA, indicating that PrP surface exposure is required for executing this effect. Proteins that in their native forms were not responsive to PS-DNA, such as thymocyte antigen 1 (Thy1), Doppel protein (Dpl), green fluorescent protein (GFP), and cyan fluorescent protein (CFP), became susceptible to PS-DNA-mediated down-regulation following introduction of the N terminus of PrP into their sequence. These observations demonstrate the essential role of the N-terminal domain for promoting oligonucleotide-mediated reduction of the PrP level and suggest that transient treatment of cultured cells with PS-DNA may provide a general method for targeted modulation of the levels of desired surface proteins in a conditional and reversible manner.

Phosphorothioate oligonucleotides reduce PrP levels and prion infectivity in cultured cells.

Prions are composed solely of the disease-causing prion protein (PrPSc) that is formed from the cellular isoform PrPC by a posttranslational process. Here we report that short phosphorothioate DNA (PS-DNA) oligonucleotides diminished the levels of both PrPC and PrPSc in prion-infected neuroblastoma (ScN2a) cells. The effect of PS-DNA on PrP levels was independent of the nucleotide sequence. The effective concentration (EC50) of PS-DNA required to achieve half-maximal diminution of PrPSc was approximately 70 nM, whereas the EC50 of PS-DNA for PrPC was more than 50-fold greater. This finding indicated that diminished levels of PrPSc after exposure to PS-DNA are unlikely to be due to decreased PrPC levels. Bioassays in transgenic mice demonstrated a substantial diminution in the prion infectivity after ScN2a cells were exposed to PS-DNAs. Whether PS-DNA will be useful in the treatment of prion disease in people or livestock remains to be established.