Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins
Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins
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DOI：
10.1016/j.jviromet.2008.03.013
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发表时间：
2008-07-01
影响因子：
3.1
通讯作者：
Garson, Jeremy A.
中科院分区：
文献类型：
--
作者：
Bibby, David F.;Gill, Andrew C.;Garson, Jeremy A.
传染性海绵状脑病的临床诊断和研究受到缺乏足够敏感和特异性试剂的阻碍，这些试剂能够充分检测朊病毒蛋白PrPC和病理亚型PrPSc的正常细胞形式。为了提供这样的试剂，我们应用针对重组鼠朊病毒蛋白的指数富集配体系统进化（SELEX）来选择高亲和力的单链DNA配体（适体）。SELEX协议和随后的适体表征采用蛋白质固定/分区使用镍络合的磁性颗粒和一种新的SYBR Green介导的定量实时PCR技术。在8轮选择后，克隆富集的适体库，并对24个克隆进行测序。其中七个是“孤儿”克隆人，其余的被分成三个独立的T丰富的家庭。除了四个适体克隆外，所有适体克隆都表现出与鼠朊病毒蛋白的特异性结合，并且大多数适体克隆还与人和羊朊病毒蛋白结合。解离常数（Kd）范围为18至79 nM。流式细胞术与荧光素标记的适配体证实，结合细胞依赖于表达的PrPc。初步研究还表明，三价适体池能够结合胍变性后的病理亚型PrPSc。(C)2008 Elsevier B. V.保留所有权利。
Clinical diagnosis and research into transmissible spongiform encephalopathies are hampered by the lack of sufficiently sensitive and specific reagents able to adequately detect the normal cellular form of the prion protein, PrPC, and the pathological isoform, PrPSc. In order to provide such reagents, we applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against a recombinant murine prion protein, to select single-stranded DNA ligands (aptamers) of high affinity. The SELEX protocol and subsequent aptamer characterisation employed protein immobilisation/partitioning using nickel-complexed magnetic particles and a novel SYBR Green-mediated quantitative real-time PCR technique. Following eight rounds of selection, the enriched aptamer pool was cloned and 24 clones sequenced. Seven of these were 'orphan' clones and the remainder were grouped into three separate T-rich families. All but four of the aptamer clones exhibited specific binding to the murine prion protein and the majority also bound to human and ovine prion proteins. Dissociation constants (K-d) ranged from 18 to 79 nM. Flow cytometry with fluorescein-labelled aptamers confirmed that binding to cells was dependent on the expression of PrPc. Preliminary studies also indicate that a trivalent aptamer pool is capable of binding the pathological isoform PrPSc following guanidinium denaturation. (C) 2008 Elsevier B.V. All rights reserved.