Arnold, Steve and Missailidis, Sotiris
Development of aptamers that bind to BACE1.
Alzheimer's & Dementia, 5(4 Sup.) P416-P416.
Background: The Beta-secretase amyloid cleaving enzyme (BACE1) is the primary enzyme involved in the processing of amyloid precursor protein (APP), releasing the amyloidgenic polypeptides that aggregate to form senile plaques observed on micrographs ofADbrains. Thus, inhibition of BACE1 is expected to avoid or at least slow down further formation of insoluble deposits. Aptamers are small synthetic DNA or RNA oligonucleotides with the ability to bind to proteins and enzymes in a highly specific manner. Aptamers raised against the beta secretase have the potential to interfere with the normal amyloid precursor protein - beta secretase interactions, thus preventing plaque formation and lead to the reduction of neuro-toxic amyloid and the arresting of beta-amyloid toxicity. Methods: Variations in the SELEX methodology have been used for aptamer selection against BACE1 from DNA single stranded libraries. The library was composed of a 25base long variable region, flanked by primers for PCR amplification. Selection was performed on ELISA plates and microcon filters, followed by cloning and sequencing. Characterisation of aptamer-BACE1 interactions was performed using EMSA, ELISA and fluorescent based inhibition assays. Results: A number of SELEX protocols were used to identify aptamers against this enzyme, as aptamer selection against BACE1 presented significant difficulties. These, however, were mainly separated in two categories, one focused around the enzyme immobilised on ELISA plates and challenged with the aptamer library, and one focused on solution-based selections using microcon filters of appropriate molecular weight cut-off and elution using high salt. Selected aptamers were found to be in a stem and loop conformation, calculated using RNA Shapes, and were shown to bind to the target enzyme, as judged by EMSA and fluorescence studies, as well as by antibody competition studies in ELISA type assays. Conclusions: Aptamers have been selected against the BACE1 enzyme and have a confirmed binding of high affinity and specificity. Thus, aptamers offer the possibility for development of novel inhibitors in AD, both as therapeutic entities and, if appropriately labelled with MRI or radiolabels, as novel imaging agents.
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