Victor Adler, Brian Zeiler, Valentin Kryukov, Richard Kascsak, Richard Rubenstein and Abraham Grossman
1 Q-RNA Inc., 3960 Broadway, New York, NY 10032, USA
2 Institute for Basic Research in Developmental Disabilities, 1050
Forest Hill Road, Staten Island, NY 10314, USA
e-mail: grossman@q-rna.com
We have identified a small, highly structured (shs)RNA that binds human recombinant prion protein (hrPrP) with high affinity and specificity under physiological conditions (e.g. 10% bovine calf serum (BCS), neutral pH, nanomolar concentrations of RNA and hrPrP). We also demonstrate the ability of this shsRNA to form highly stable nucleoprotein complexes with hrPrP and cellular PrP (PrPC) from various cell extracts and mammalian brain homogenates. The apparent mass of the nucleoprotein complex is dependent on the molar ratio of hrPrP to RNA during complex formation. The hrPrP in these complexes acquires resistance to degradation by Proteinase K (PK). Other shsRNAs, however, under identical conditions, neither form stable complexes with hrPrP nor do they induce resistance to PK digestion. We also demonstrate that the RNAs in these nucleoprotein complexes become resistant to ribonuclease A hydrolysis. These interactions between shsRNAs and hrPrP suggest possible roles of RNAs in the modulation of PrP structure and perhaps disease development. ShsRNAs that bind to hrPrP with high affinity and induce resistance to PK digestion can be used to develop molecular biology assays for the screening of compounds associated with PrP structure transformation or for drugs that inhibit this process.
Author Keywords: RNA structure; prion; PrP; RNA–protein interactions; BSE
Abbreviations: PK, Proteinase K; shsRNAs, small, highly structured
RNAs; MBE, mouse brain
extract; PrPSen, PrP sensitive to PK digestion;
PrPRes, pK resistant PrP; PrPC, cellular isoform of
PrP; PrPSc, scrapie isoform of PrP; pmol, picomoles;
EM, electron microscopy; RNase, A,
ribonuclease A; nt, nucleotide(s); TSE, transmissible
spongiform encephalitis; PAGE,
polyacrylamide gel electrophoresis; wt, wild-type
1. Frenster JH, "Nuclear RNA Species Activate DNA Transcription within Chromatin".
2. Gottesfeld JM, and Barbas CF III, "RNA as a Transcriptional Activator".
3. Hovsepian JA, and Frenster JH, "RNA-Induced Melting of DNA during Selective Gene Transcription".
4. Saha S, Ansari AZ, Jarell KA, and Ptashne M, "RNA Sequences that Work as Transcriptional Activating Regions".
5. Frenster JH, "Ultrastructural Probes of Active
DNA Sites, and the RNA Activators of DNA".