Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

Abstract

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2 -fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrP Sc than for recombinant PrP C and inhibited the accumulation of PrP res in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from nonspecific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, ␣-helix-rich and ␤-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the ␤-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the ␣-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the ␣-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is ␤-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.

Human prions and plasma lipoproteins

“…The affinity of native PrP Sc for apoB appears to be much greater than that of any PrP Sc -specific antibody or aptamer reported to date (50)(51)(52)(53). The binding of native PrP Sc to apoB was enhanced up to 10-fold by the additional lipids found in LDLs (Table 1).…”

Methionine 129 Variant of Human Prion Protein Oligomerizes More Rapidly than the Valine 129 Variant

“…There is no evidence so far that these ␤-oligomers exist in vivo or can directly cause prion diseases either alone or with the help of other factors. The high affinity and newly reported ligands that we have isolated in our laboratory (50,51) would help investigating the presence in vivo of the ␤-oligomers. The observed differences in the misfolding behavior between Met 129 and Val 129 variants could provide a kinetic explanation to the observed high susceptibility of individuals that are methionine homozygote to sporadic as well as to variant CJD.…”

Anti-bovine Prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity

“…Aptamers are artificial nucleotides derived from systematic evolution of ligands by exponential enrichment (SELEX), 6,7 which bind a wide range of targets with a high affinity and specificity, as is seen with antibodies. 8,9 Using this method, many aptamers specific to PrP [10][11][12][13][14][15][16][17][18][19][20] and PrP Sc , 12,13 have been isolated.…”