Characterization and application of a novel RNA aptamer against the mouse prion protein.
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 (K(d) = 5.6 +/- 1.5 nM), and 2'-fluoro-pyrimidine modifications for RNase resistance did not abolish its binding activity (K(d) = 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.
Sekiya
Satoru
S
Cooperative Graduate School, University of Tsukuba, Tennodai, Tsukuba, Ibaraki.
Noda
Ken
K
Nishikawa
Fumiko
F
Yokoyama
Takashi
T
Kumar
Penmetcha K R
PK
Nishikawa
Satoshi
S
D000595:Amino Acid Sequence; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
D052157:Aptamers, Nucleotide; D010446:Peptide Fragments; D011328:Prions; D012313:RNA