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.

中文翻译：

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含有串联 GGA 重复序列的抗牛朊病毒蛋白 RNA 适体与重组牛朊病毒蛋白及其β 异构体以高亲和力相互作用。

为了获得针对牛朊病毒蛋白 (bPrP) 的 RNA 适配体，我们从包含 55 个核苷酸的随机区域的 RNA 库中进行了体外选择，以靶向重组 bPrP。大多数获得的适体包含保守的 GGA 串联重复序列 (GGA)(4) 和适体 #1 (apt #1) 对 bPrP 及其 beta 异构体 (bPrP-beta) 显示出高亲和力。apt #1 的序列表明它具有 G-四链体结构，这在用 KCl 滴定时使用 CD 光谱得到证实。该保守区域的诱变研究和竞争性分析表明，保守 (GGA)(4) 序列对于与 bPrP 和 bPrP-β 的特异性结合很重要。在 5'-生物素化之后，适配体 #1 在西北印迹试验中专门检测到牛脑匀浆中的 PrP(c)。蛋白质缺失突​​变体分析显示 bPrP 适体结合 bPrP 序列的 25-131。有趣的是，与 apt #1 相比，最小化的适体 #1 (17 nt) 显示出对 bPrP 和 bPrP-beta 的更大结合。因此，这种最小化形式的适配体#1 可用于检测 bPrP、诊断朊病毒疾病、富集 bPr 并最终更好地了解朊病毒疾病。