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Transmembrane molecular machines

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Watson2015.pdf (10.97Mb)
Date
30/06/2015
Author
Watson, Matthew A.
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Abstract
Transmembrane molecular machines are ubiquitous in nature. These evolved systems demonstrate superlative elegance and efficiency of operation. Imitation and hijacking of biological components such as proteins and DNA has emerged as a means of imparting desirable characteristics to rationally designed synthetic molecular machines. This thesis presents work towards various synthetic transmembrane molecular machines based on alpha-haemolysin (α-HL). Chapter One reviews progress towards synthetic transmembrane machines, introducing natural examples, defining criteria for being ‘a molecular machine’ cataloguing examples and trends in synthetic molecular machines in solution, on surfaces and in membranes. Examples are evaluated in terms of their machine like behaviour and α-HL emerges as a particularly promising component in the development of synthetic transmembrane molecular machines. Chapter Two examines solvent isotope effects resulting from substitution of hydrogen by deuterium in water at the nanoscale – on the rates of transmembrane ion transport and transmembrane translocation of ssDNA through α-HL, both of which are of concern in the context of building molecular machines which use α-HL as a component. Chapters Three to Six look at different machine applications of related transmembrane architectures based on individual transmembrane rotaxanes constructed in α-HL from DNA/PEG copolymer ‘thread’ strands and DNA ‘primer’ strands. Chapter Three uses this approach to observe translational motion of the thread strand in both directions along the z-axis due to nucleotide incorporation and pyrophosphorolysis in real-time with single-nucleotide resolution. Chapter Four provides the first demonstration of asymmetrical, hysteretic cyclical behaviour in the translational motion of the thread strand by incorporation of a nicking site which resets the system after nucleotide incorporations have occurred. Chapter Five introduces a novel variant of the rotaxane architecture using a circularised primer strand which allows real time observation of rolling circle amplification at the single molecule level by coupling the process to the unidirectional translocation of the thread strand. Chapter Six considers the use of the vestibule of α-HL as a transmembrane DNA ligase mimic with the DNA thread/primer complex as substrate.
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https://hdl.handle.net/1842/39361

http://dx.doi.org/10.7488/era/2611
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  • Chemistry thesis and dissertation collection

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