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Journal of Biochemistry 2006 139(3):383-390; doi:10.1093/jb/mvj046
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© 2006 The Japanese Biochemical Society.

Regular Paper

Characterization and Application of a Novel RNA Aptamer against the Mouse Prion Protein

Satoru Sekiya1,2, Ken Noda3, Fumiko Nishikawa4, Takashi Yokoyama5, Penmetcha K.R. Kumar4 and Satoshi Nishikawa1,2,*

1 Cooperative Graduate School, University of Tsukuba, Tennodai, Tsukuba, Ibaraki; 2 Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki; 3 National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry & Fisheries, Kokubunji, Tokyo; 4 Institute for Biological Resources and Functions, AIST, Tsukuba, Ibaraki; and 5 Prion Disease Research Center, National Institute of Animal Health (NIAH), Tsukuba, Ibaraki

* To whom correspondence should be addressed at: Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Higashi, Tsukuba, Ibaraki 305–8566. Tel: +81-29-861-6097, Fax: +81-29-861-6095, E-mail: satoshi-nishikawa{at}aist.go.jp

In order to isolate RNA aptamers against the mouse prion protein (mPrP), we carried out in vitro selection from RNA pools containing a 30-nucleotide randomized region. Aptamer 60-3 was found to have a high affinity for mPrP (Kd = 5.6 ± 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (Kd = 22 ± 4 nM). Following 5' biotinylation, aptamer 60-3 specifically detected PrP in mouse brain homogenate in a Northwestern blotting assay. To determine the mPrP–aptamer binding region, we performed protein-deletion-mutant analysis and competition-binding analysis using heparin. The results showed that aptamer 60-3 appears to have binding sites located between amino acids 23–108.


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