In Vitro Selection And Characterization Of DNA Aptamers Target To β-amyloid42(Aβ42)

Alzheimer’s disease (AD), whose cause is not clear, is a neurodegenerativedisease. The pathological hallmarks of AD are amyloid plaques (AP) andneurofibrillary tangles (NFT). So far, there is still no effective therapeutic method forAD. So it is very important to diagnose AD in the early stage for prevention from AD.Numerous studies have demonstrated that beta-amyloid (A) proteins, which tend toaggregate and deposit as amyloid plaques in the brain, play a key role in thepathogenesis of AD. Beta-amyloid42(A42), which is one of the major isoforms ofA is more pathogenic than Aβ40, although the level of Aβ40in cerebrospinal fluidhas been found to be much higher than that of A42. Morever, A42has beenestablished as a biomaker for AD. So it is essential to develop ligands specificallybinding to A42.As a kind of new molecular recognition ligands, aptamers have attractedextensive attention because of their various unique features. Aptamers have beenwidely used in biological medicine, clinical treatment, as well as biosensing. Theyhave the characteristics of a wide range of targets, easy production and modificati on，high stability and affinity as well as good specificity. However, the applications ofaptamers in AD diagnosis and therapy have been restricted by the lack of aptamersfor A42. So it is essential to screen aptamers aganist A42for the development ofAD diagnosis and therapy.This thesis focused on applying library immobilization SELEX strategy to selectaptamer target to A42. The selected DNA sequences were also identified. The mainpoints of this thesis are summarized as follows:(1) Aptamer selectionThe DNA library was fixed on streptavidin coated beads by means ofhybridization with biotinylated antisense strands. Then, the target was incubated withthe immobilized library. At last, the ssDNA which combined with the target would becollected after speraration. Ten rounds screening were carried out, and the librar y ofthe seventh round and the ninth round were enriched and then sent for cloning andsequencing. Three selected DNA sequences were picked out for the furtherexperiments after sequencing result analysis.(2) Characterization of the selected sequences The results of the affinity accessment, and specificity investigation indicatedthat two aptamers named A7-92and A9-103with high affinity and specificity toA were obtained. In addition, in order to make the aptamers more applicable forapplication, we further optimized the two sequences and verified its targetrecognition capacity. The experimental results showed that there were four truncatedaptamers which recognized A and the truncated aptamer, named A7-92-1,showed comparable affinity to A with A7-92-1. Notably, all aptamers had thespecificity to A42, allowing the discrimination of A42from A40, although theonly defference between A42and A40is the additional residues at the C-terminalof A42. At last, we evaluated whether the aptamers selected here had the ability toprevent A42aggregation.The results of dynamic light scattering (DLS) and atomicforce microscopy (AFM) demonstated that the aptamer A7-92-1could prevent A42aggregation to some extent.