Selection of aptamers for amyloid beta-protein, the causative agent of Alzheimer's disease.
Selection of aptamers for amyloid beta-protein, the causative agent of Alzheimer's disease.
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DOI：
10.3791/1955
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发表时间：
2010-05
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影响因子：
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通讯作者：
F. Rahimi;G. Bitan
中科院分区：
文献类型：
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作者：
F. Rahimi;G. Bitan
阿尔茨海默病(AD)是一种进行性的、与年龄相关的神经退行性疾病，其隐蔽的病程使其症状前诊断变得困难(1)。明确的AD诊断只有在死后才能实现，因此建立症状前的早期诊断对开发和实施有效的治疗方法至关重要(2，3)。淀粉样β蛋白(Abeta)在AD发病机制中起核心作用。可溶性的寡聚体Abeta组件被认为影响AD患者突触功能障碍和神经元丢失的神经毒性(4，5)。已经描述了各种形式的可溶性Abeta组件，然而，它们与AD病因和发病机制的相互关系和相关性是复杂的，并且还不是很清楚。特定的分子识别工具可以解开Abeta组件之间的关系，并有助于在症状出现之前在疾病过程的早期检测和表征这些组件。分子识别通常依赖于抗体。然而，另一类分子识别工具，适配子，提供了相对于抗体的重要优势(7，8)。适配子是通过体外选择产生的寡核苷酸：通过指数浓缩的配体的系统进化(SELEX)(9，10)。SELEX是一个迭代过程，类似于达尔文进化，允许选择、扩增、丰富和永久保留某种性质，例如亲和性、特异性、配体结合(适配子)或催化活性(核酶和DNA酶)。尽管适配子在现代生物技术和医学中作为工具出现(11)，但它们在淀粉样蛋白领域一直没有得到充分利用。针对不同形式的PrP(PrP)(12-16)，很少有RNA或ssDNA适配子被选择。针对重组牛PrP产生的RNA适配子被证明能够识别牛PrP-β(17)，这是一种形成淀粉样纤维(18)的全长PrP的可溶性、寡聚体、富含β片层的构象变体。使用单体和几种形式的纤维β(2；)-微球蛋白(β(2；)m)产生的适配子被发现与除β(2；)m纤维(19)之外的某些其他淀粉样蛋白的纤维结合。Ylera等人。描述了针对固定化单体Abeta40(20)选择的RNA适配子。出乎意料的是，这些适配子结合了纤维蛋白Abeta40。总之，这些数据提出了几个重要的问题。为什么针对单体蛋白选择的适配子识别它们的聚合形式？能否获得针对淀粉样蛋白单体和/或寡聚体形式的适配子？为了解决这些问题，我们试图为用光诱导未修饰蛋白(PICUP)(22，23)产生的共价稳定的寡聚体Abeta40(21)选择适配子。与之前的发现(17，19，20)相似，这些适配子与Abeta的纤维和其他几种可能识别潜在常见的淀粉样蛋白结构的淀粉样蛋白发生反应(21)。在这里，我们介绍了在生产这些适配子(21)中使用的SELEX方法。
Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult(1). Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies(2,3). Amyloid beta-protein (Abeta) is central to AD pathogenesis. Soluble, oligomeric Abeta assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD(4,5). Various forms of soluble Abeta assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood(6). Specific molecular recognition tools may unravel the relationships amongst Abeta assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies(7,8). Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)(9,10). SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes). Despite emergence of aptamers as tools in modern biotechnology and medicine(11), they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)(12-16). An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-beta(17), a soluble, oligomeric, beta-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils(18). Aptamers generated using monomeric and several forms of fibrillar beta(2;)-microglobulin (beta(2;)m) were found to bind fibrils of certain other amyloidogenic proteins besides beta(2;)m fibrils(19). Ylera et al. described RNA aptamers selected against immobilized monomeric Abeta40(20). Unexpectedly, these aptamers bound fibrillar Abeta40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric Abeta40(21) generated using photo-induced cross-linking of unmodified proteins (PICUP)(22,23). Similar to previous findings(17,19,20), these aptamers reacted with fibrils of Abeta and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope(21). Here, we present the SELEX methodology used in production of these aptamers(21).