Selection of RNA Aptamers and Their Recognition of Amyloid Assemblies
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Selection of RNA aptamers and their recognition of amyloid assemblies Rachel Alexandra Mahood Submitted in accordance with the requirements for the degree of Doctor of Philosophy The University of Leeds Astbury Centre for Structural Molecular Biology September 2015 The candidate confirms that the submitted work is her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated overleaf. The candidate confirms the appropriate credit has been given within the thesis where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from this thesis may be published without proper acknowledgement. © 2015 The University of Leeds and Rachel Alexandra Mahood Acknowledgements It is with great pleasure that I thank all the people who have helped me throughout my time in Leeds and who have made the completion of my PhD one of the most rewarding experiences of my life. Firstly, to my supervisors, Prof. Sheena Radford and Prof. Peter Stockley. This work would have been impossible without your constant support, guidance and unwavering enthusiasm. I’m incredibly grateful for the time and hard work that has gone into all aspects of the project, and in preparing this thesis. Extra special thanks go to Sheena, for knowing exactly what to say during the toughest of times, and never allowing me to give up. I’d also like to express my deepest gratitude to all members of the Radford and Stockley laboratories, past and present, who have provided endless advice and support during this project, but most importantly, kept me smiling. In particular, David Bunka, Claire Sarell and Simon White, who all understand the joys of amyloid and aptamers, and dedicated a huge amount of time to helping me with all aspects of the work. Thanks to Maya Pandya, Katie Stewart and Tiernan O’Malley for the help with the Aβ prep. Thanks also to Eric Dykeman, for his work on the bioinformatics, James Ault for the mass spectrometry and Matthew Jackson for all things α-syn (and the laughs along the way!). I’d also like to thank our wonderful lab manager Nasir Khan. Nothing is ever too much to ask and I’m so grateful for your constant help and the endless supply of biscuits! The lab is a much more pleasant place to work because of you. Huge thanks go to two of my favourite ladies, Lydia Young and Jan Saunders (co-founder of the stagnant puddle). It was such a pleasure, not only to collaborate with you two on the amazing work you did, but also to have you as such incredible friends. Jan, your help in the final few months (and throughout – I’ll never forget the madness of our little corner!) was nothing short of amazing and I couldn’t have done it without you. Special thanks also go to Claire, Sasha, Sophie and Helen; for all the sneaky Costa breaks and cocktails, and making the last four years so much fun. Extra special thanks go to my Leeds wifey, Nic, for being such a fantastic housemate for nearly 7 years! I have so many wonderful memories of our time in Leeds and will be very sad to leave. Finally, I would like to thank all my lovely family, especially Mum and Dad; I certainly couldn’t have done this without your love and encouragement. And Joe, thank you for being so patient and putting up with me – knowing I had you to come home to in the end kept me going, and I hope I’ve made you proud. i ii Abstract 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. iii iv Table of Contents ACKNOWLEDGEMENTS .......................................................................................................... I ABSTRACT ............................................................................................................................... III TABLE OF CONTENTS ............................................................................................................ V LIST OF FIGURES ................................................................................................................ XIII LIST OF TABLES ................................................................................................................. XVII LIST OF ABBREVIATIONS ................................................................................................. XIX LIST OF AMINO ACID ABBREVIATIONS .................................................................. XXVII 1 INTRODUCTION ............................................................................................................... 1 1.1 Amyloid disease: A modern epidemic ....................................................................................... 1 1.2 Protein misfolding and aggregation .......................................................................................... 2 1.2.1 Principles of protein folding and misfolding ............................................................................... 2 1.2.2 Cellular response to aggregation ................................................................................................ 5 1.2.3 Mechanism of amyloid formation .............................................................................................. 8 1.2.4 Structure of amyloid fibrils ....................................................................................................... 10 1.2.5 Other structures of the amyloidogenic pathway ...................................................................... 13 1.2.6 Amyloid formation and disease ................................................................................................ 15 1.2.7 Therapeutic approaches to amyloid diseases ........................................................................... 19 1.2.7.1 Inhibiting the production of the amyloid precursor ........................................................ 19 1.2.7.2 Stabilising the native protein ........................................................................................... 20 1.2.7.3 Inhibition of the formation of toxic aggregates ............................................................... 20 1.2.7.4 Accelerating fibril formation ............................................................................................ 23 1.2.7.5 Stimulating aggregate degradation.................................................................................. 23 1.3 Alzheimer’s disease and Aβ .....................................................................................................25 1.3.1 Alzheimer’s disease .................................................................................................................. 25 1.3.2 The Aβ peptide ......................................................................................................................... 26 v 1.3.3 The amyloid cascade hypothesis ...............................................................................................28 1.3.4 Neurotoxic