Amyloidoses are a group of protein misfolding disorders which are characterised by the abnormal accumulation of highly ordered filamentous assemblies, known as amyloid fibrils. More than 50 human disease states are attributed to this phenomenon, many of which are neurodegenerative and pose an ever-increasing threat to our aging society. There is a clear need to dissect the processes behind such disorders, as well as to provide novel and much needed treatments and diagnostic reagents. This thesis was motivated by the recognition powers of RNA molecules, which can be discovered through the in vitro selection of RNA aptamers. RNA aptamers are a well-established class of research tools, imaging probes, diagnostic reagents and therapeutics, which are showing increasing promise in many fields, but are currently not exploited in the detection or treatment of amyloid disorders.
In this thesis, the recognition power of RNA aptamers was explored in targeting species associated with the most prevalent and deadly amyloid disorder, Alzheimer’s disease. In vitro selection of RNA aptamers was performed against immobilised monomeric Aβ40, as well as two structurally distinct Aβ40 amyloid fibrils, formed in vitro. The anti-monomer aptamers were unable to recognise native, monomeric Aβ40 in solution but instead displayed enhanced affinity for the fibrillar forms. Using fluorescence polarisation, the anti-fibril aptamers have been shown able to cross-react with other fibril polymorphs, formed from both Aβ40 and the unrelated protein sequence, α-synuclein, indicating generic amyloid selectivity. Aptamers were, however, unable to recognise amyloid fibrils assembled from short amyloidogenic peptides. Amyloid recognition by aptamers was shown to be largely independent of the RNA sequence or structure. Further structural characterisation, including competition experiments with generic amyloid-binding molecules glycosaminoglycans, indicated that binding was most likely mediated through the phosphate backbone contacts with ordered repeats of positively charged regions of the amyloid assemblies.
Overall, the results demonstrate an inherent affinity for amyloid by RNA molecules, making it highly challenging to select aptamers able to distinguish between different cross-β assemblies. However, the seemingly universal amyloid-binding propensity demonstrated by RNA could allow development of generic amyloid detection tools, more effective than current methods. Furthermore, the work indicates the need to explore possible roles of RNA associations as a general amyloid toxicity mechanism.
