Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins

Bibby, David F. and Gill, Andrew and Kirby, Louise and Farquhar, Christine F. and Bruce, Moira E. and Garson, Jeremy A. (2008) Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins. Journal of Virological Methods, 151 (1). pp. 107-115. ISSN 0166-0934

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Abstract

Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrPC, and the pathological isoform, PrPSc. In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were ‘orphan’ clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (Kd) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrPC. Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrPSc following guanidinium denaturation.

Additional Information:The final published version of this article can be accessed online at https://www.sciencedirect.com/science/article/pii/S0166093408001018?via%3Dihub
Keywords:prion protein, DNA aptamer, SELEX, transmissible spongiform encephalopathies, PrPSc
Subjects:C Biological Sciences > C760 Biomolecular Science
Divisions:College of Science
ID Code:29556
Deposited On:25 Oct 2018 14:15

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