DNA Aptamers That Bind to PrP^C and Not Prp^Sc Show Sequence and Structure Specificity

Abstract

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP(C)) 23-231 by systematic evolution of ligands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 10(24) distinct nucleic acid species. Sixty nanograms of rhuPrP(C)23-231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrP(C)23-231 at 10(-6) M to 10(-8) M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP(C)90-231. Theoretical thermodynamic modeling of selected aptamer sequences identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrP(C)23-231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrP(C) expressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrP(C) specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrP(C) and mammalian PrP(C) with varying affinities and can be applied to biological samples for PrP(C) enrichment and as diagnostic tools in double ligand assay systems.

Prion Protein Complexed to N2a Cellular RNAs through Its N-terminal Domain Forms Aggregates and Is Toxic to Murine Neuroblastoma Cells

“…Questions remain unanswered, however, regarding the size and structure of the RNA that are necessary for interaction with PrP C and for catalysis of this conversion. In light of these results, we investigated the interaction of prokaryotic and eukaryotic RNA extracts and of two synthetic RNAs (SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] and 18-mer opRNA) ( Fig. 1 and supplemental Table S1) with rPrP23-231 and with two N-terminal rPrP deletion mutants (rPrP⌬51-90 and rPrP⌬32-121).…”

“…High pressure liquid chromatography-purified synthetic single-stranded RNA fragment 43-59 of SAF93 aptamer (SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] ) (22) and opRNA, a random 18-mer sequence, derived from a translational operator of MS2 bacteriophage (37) were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Nucleotide sequences were: SAF93 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] , 5Ј-GGA UGC AAU CUC CAU CCC-3Ј; and opRNA, 5Ј-AAA CAU GGG UUC CCA UGU-3Ј. Synthetic RNA samples were maintained lyophilized at Ϫ20°C and used in RNase-free water.…”

“…The spectra were processed using TOPSPIN 2.1 (Bruker) and analyzed with CARA 1.8 (38 [43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59] . Transmission Electron Microscopy-Samples were adhered to a carbon-coated grid, blotted to remove excess material, and stained for 10 s with a 2% solution of uranyl acetate prepared in water.…”

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

“…16 Takemura et al proposed that PrP Sc -enrichment with pre-treatment using PrP Cspecific aptamers, to remove normal prions from a sample, could be applied as diagnostic tools in double ligand assay systems. 17 Such an To prepare C#1, the generated PCR fragment using plasmid #1 as template and the proper primers (+) c#1 and (-) c#1, were used for in vitro transcription by T7 RNA polymerase as described above. Binding assay of anti-bPrP aptamer.…”

Hemin Interactions and Alterations of the Subcellular Localization of Prion Protein

“…N2aGFP-GPI is an N2a cell line that was stably transfected with a GFP-GPI (glycosylphosphatidylinositol-anchored green fluorescent protein) expression vector (26). CF10 cells generated from PrP C null mice (27) were transduced with a murine retroviral vector (pSFF) encoding full-length hamster PrP C or hamster PrP C lacking residues 34 -94 (28). Human neuroblastoma cells (NB1) express endogenous levels of PrP C .…”