title: Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its beta isoform with high affinity.
abstract: In order to obtain RNA aptamers against bovine prion protein (bPrP), we carried out in vitro selection from RNA pools containing a 55-nucleotide randomized region to target recombinant bPrP. Most of obtained aptamers contained conserved GGA tandem repeats (GGA)(4) and aptamer #1 (apt #1) showed a high affinity for both bPrP and its beta isoform (bPrP-beta). The sequence of apt #1 suggested that it would have a G-quadruplex structure, which was confirmed using CD spectra in titration with KCl. A mutagenic study of this conserved region, and competitive assays, showed that the conserved (GGA)(4) sequence is important for specific binding to bPrP and bPrP-beta. Following 5'-biotinylation, aptamer #1 specifically detected PrP(c) in bovine brain homogenate in a Northwestern blotting assay. Protein deletion mutant analysis showed that the bPrP aptamer binds within 25-131 of the bPrP sequence. Interestingly, the minimized aptamer #1 (17 nt) showed greater binding to bPrP and bPrP-beta as compared to apt #1. This minimized form of aptamer #1 may therefore be useful in the detection of bPrP, diagnosis of prion disease, enrichment of bPr and ultimately in gaining a better understanding of prion diseases.
authors.affiliation: Graduate School of Life and Environmental Science, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Murakami
authors.forename: Fumiko
authors.initials: F
authors.lastname: Nishikawa
authors.forename: Ken
authors.initials: K
authors.lastname: Noda
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nishikawa
mesh_terms: D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002417:Cattle; D018621:PrPC Proteins / Q000737:chemistry* / Q000378:metabolism; D017096:Prion Diseases / Q000175:diagnosis / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018911:Trinucleotide Repeats
chemical_list: D052157:Aptamers, Nucleotide; D018621:PrPC Proteins; D011994:Recombinant Proteins