In vitro selection of anti-mouse prion protein RNA aptamers
Satoru Sekiya^'^, Ken Noda^, Penmetcha K.R. Kumar^, Takashi Yoko- yama^ and Satoshi Nishikawa^'^
^Graduate School of Life and Environmental Sciences, University of Tsu- kuba ^National Institute for Biological Resources and Functions, Na- tional Institute of Advanced Industrial Science and Technology (AIST),
1-1-1 Higashi, Tsukuba 305-8577 Japan ^Nationl Veterinary Assay Laboratory (NVAL) "^Prion Disease Research Center, National Institute of Animal Health (NIAH) <e-mail> s2-sekiya@aist.go.jp
Abstract
Since the in vitro selection method, which chooses nucleic acid molecules of demands from huge scale clot of randomized nucleic acid molecules, has been developed (Tuerk, et al.. Science.; 249 (4968):505-10, 1990 etc.), various species of functional nucleic acid molecules have been developed.
Notably, one of those molecules called "Aptamer" can specifically recog- nizes and binds to its target from small molecule such as amino acid to large molecules such as proteins or virus particles. As its property is al- most same as antibody, aptamer has been applied in basic study of nucleic acid/protein interaction and clinical or diagnostic drugs. (Allen, et al..
Virology, 209, 327-336, 1995 etc.)
Recently, screening of anti-prion RNA aptamers have been carried on, expecting for the application to the prion diseases diagnostics or therapeu- tics, and novel tools for studying PrP^"^ generation mechanism. In order to apply for diagnostics, we aimed to isolate anti-mouse prion protein (anti-mPrPc) aptamer.
From artificial RNA library including 30 mer random region, we carried out in vitro selection cycles and concentrated the candidate RNA mole- cules. After 10 cycles of selection, we obtained three types of anti-mPrPc RNA aptamers. These aptamers have high affinity to mPrPc (Kd= 10 nM), and 2'-fluoro modification for RNase resistance did not interrupt their binding to mPrPc (Kd= 20 nM). These aptamers mainly bind to N-terminus region of mPrPc including octa-repeat (amino acid 23-230), but also bind to C-terminus region (amino acid 89-230) weakly. Fur-
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