DNA aptamers that bind to PrP^C and not PrP^Sc show sequence and structure specificity

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 ^C23-231 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^C23-231 at 10 ^-6 M to 10^-8 M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrP^C90-231. 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^C23-231 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.