Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its beta isoform with high affinity.
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.
Murakami
Kazuyoshi
K
Graduate School of Life and Environmental Science, University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan.
Nishikawa
Fumiko
F
Noda
Ken
K
Yokoyama
Takashi
T
Nishikawa
Satoshi
S
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
D052157:Aptamers, Nucleotide; D018621:PrPC Proteins; D011994:Recombinant Proteins