Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease

Abstract

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).