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ORIGINAL RESEARCH ARTICLE

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

Kaori Takemura^*, Ping Wang^*, Ina Vorberg, Witold Surewicz, Suzette A. Priola, Anumantha Kanthasamy, Ravi Pottathil^||, Shu G. Chen^¶ and Srinand Sreevatsan^*,1

^* Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, the Ohio State University, Wooster, Ohio 44691; Laboratory of Persistent Viral Infections, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana 59840; Department of Physiology and Biophysics; Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011; ^|| AccuDx Inc., San Diego, California 92126; and ^¶ Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106

To whom requests for reprints should be addressed at ¹ Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, 1365 Gortner Avenue, 225 VTH, St. Paul, MN 55108. E-mail: sreev001{at}umn.edu

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 10²⁴ 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.

Key Words: DNA aptamers • prion • scrapie • TSE

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