In:
Experimental Biology and Medicine, SAGE Publications, Vol. 231, No. 2 ( 2006-02), p. 204-214
Abstract:
DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrP c ) 23–231 by systematic evolution of llgands 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.
Type of Medium:
Online Resource
ISSN:
1535-3702
,
1535-3699
DOI:
10.1177/153537020623100211
Language:
English
Publisher:
SAGE Publications
Publication Date:
2006
detail.hit.zdb_id:
2020856-X
SSG:
12
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