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 difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3. Amyloid β-protein (Aβ) is central to AD pathogenesis. Soluble, oligomeric Aβ assemblies are believed to effect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble Aβ assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst Aβ 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 antibodies7,8. 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 medicine11, 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-β17, a soluble, oligomeric, β-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar β2-microglobulin (β2m) were found to bind fibrils of certain other amyloidogenic proteins besides β2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric Aβ4020. Unexpectedly, these aptamers bound fibrillar Aβ40. 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 Aβ4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of Aβ and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Aptamers

“…(8) HFIP-treated lysozyme and IAPP contained sufficient β-sheet content as inferred from their recognition by the tested aptamers and the naïve library. Our observation of non-specific reactivity with fibrils of selected and tested aptamers, which is reminiscent of similar findings in previous studies [62,125,134,136], suggest that aptamers (and naïve libraries of oligonucleotides) likely recognize potentially common aptatopes [62,153,160].…”

“…In a recent contribution, Walter et al blended the pinching capacity of a nanomechanical DNA origami forceps [158] with the biosensing capacity of split aptamer systems [159]. Indeed, the constituting sequences of the ATP-specific split aptamer were equipped with green-and red-emitting photostable cyanine dyes that act as energy donor and acceptor, respectively [160] and were subsequently appended on one arm of the DNA origami forceps. In the absence of the analyte (i.e., ATP) the constructs remained in an open form ( Figure 5C) and emitted green light (absence of energy transfer).…”

“…Aβ fibrils and fibrils of other exemplary amyloidogenic proteins [153,160] SELES, systematic evolution of ligands by exponential enrichment; Aβ, amyloid β-protein; PICUP, photo-induced crosslinking of unmodified proteins.…”

Aptamer: A Novel Therapeutic Oligonucleotide

“…Amyloid-β made up of 36-43 amino acids is the main component of amyloid plaques found in the brain of patients with Alzheimer's disease. RNA aptamers constructed against the recombinant prion protein that forms Amyloid-β can help stop propagation of Alzheimer's disease [23]. Retroviral Integrase enzyme (HIV-1 Integrase), a potential target in HIV, found in retrovirus enables their DNA to integrate into DNA of the infected cell and thus replicate into host cells.…”

A Mathematical Analysis of Multiple-Target Selex

“…The relatively small size of aptamers allows them to penetrate tissues more effectively than antibodies, so many aptamers are being developed for detecting targets such as cancer cells, and also for detecting analytes such as blood proteins and drug concentrations, and for imaging cellular processes. Anti-VEGF 5 aptamers ( [18]) for the cure of AMD 6 and aptamers against Amyloid β−protein ( [22]) for the recognition of Alzheimer's disease are only a couple of examples of aptamers that have been developed for medical applications.…”