DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrPC) 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 rhuPrPC23-231 at 10 -6 M to 10-8 M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrPC90-231. 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 rhuPrPC23-231 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 PrPC 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 PrPC specificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrPC and mammalian PrPC with varying affinities and can be applied to biological samples for PrPC enrichment and as diagnostic tools in double ligand assay systems.
- DNA aptamers