Getting the most out of nanopores
Brinkerhoff, Henry Derek
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Over the past three decades, the fields of biophysics and biotechnology have seen an era of unprecedented growth, bolstered by the development of new experimental techniques. Prominent within these techniques are “single-molecule” methods which enable the observation and manipulation of single biomolecules. These techniques allow for controlled experiments on the most fundamental structures composing life. The function of living organisms is governed by statistical physics, and traditional bulk chemical methods report only an average of the rich and heterogeneous activity of these biological structures. Therefore, bulk methods provide an incomplete picture of the mechanical behavior of biomolecules, and of the way life stores, modifies and accesses information. Early single-molecule experiments using flow cells and magnetic tweezers were initially used to study the passive mechanics of molecules like DNA and the behav- ior of stepping enzymes including myosin and kinesin. These enzyme experiments demonstrated the power of single-molecule techniques, showing that enzyme behavior is fundamentally statistical, moving randomly with a slight rectification provided by chemical potentials maintained by the cell. Soon thereafter, an expanding repertoire of single-molecule methods including superresolution micrscopy, single-fluorophore microscopy and optical tweezers refined and expanded these results, making plain the diversity and complexity of the mechanical behavior of biomolecules. Advances in genomics over this time period made it clear that the nucleic acids DNA and RNA, which store the information passed down and used by life to encode the sequences of every protein it produces, are also subject to the statistical physics governing biomolecules. Damage to DNA and its repair, the formation of secondary structure, the insertion of viral DNA fragments, replication, recombination, modifica- tion of bases, and regulation of gene expression: these are all fundamentally random processes. As recording and analyzing vast amounts of data has become more feasible with access to greater computing power, it has become clear that methods sequencing only large samples of many DNA molecules fail to recognize the variance crucial to the functionality of life’s genetic library. The scientific appeal of a single-molecule sequencing technique together with a push for longer read lengths and cheaper sequencing led to the development of nanopore sequencing, a method using a nanometer-scale hole in a thin membrane to trap and analyze DNA. Beginning with the demonstration of nanopores as single molecule “Coulter counters,” through results proving that nanopore experiments can discriminate between trapped DNA strands with different base content, we now have arrived in an era where nanopores are used in commercial DNA sequencing platforms and high-precision single molecule biophysics experiments. Within this dissertation, I provide a “user manual” of sorts for collecting and understanding the single-molecule information provided by nanopore experiments. Then, through two examples of concrete improvements to the nanopore DNA se- quencing system, I demonstrate how a thorough understanding and adequate physi- cal model of the system can motivate experiment and invention. My hope is that a scientist wishing to perform nanopore experiments for the first time will find this to be a useful guide for executing the experiments, as well as for modeling and analyzing the rich and complex signals that they generate. In part I, background is provided on the arena in which these experiments play out. I first introduce key properties of DNA and other biological molecules, as well as the history and future of DNA sequencing. Part II contains a guide to the experimental setup and operation of a nanopore experiment. I also delve deeper into the biophysics of the experiment as it is performed at the University of Washington, discussing properties of the enzyme-DNA-nanopore complex, and I discuss the signal obtained from the experiment and its properties. In part IV, describe the ways the nanopore signal can be modeled, reduced, an- alyzed, and interpreted, including introductions to some commonplace analysis tools used to study single molecule data. Finally, part IV shows how using the results of this model, we developed extensions and modifications of the nanopore experiment, improve the accuracy and flexibility of nanopore DNA sequencing. My work at the University of Washington is included primarily in part IV, much of which I completed in collaboration with primarily Dr. Andrew Laszlo and Dr. Brian Ross, and IV, in which the variable-voltage experiments were completed in collaboration with Dr. Matthew Noakes.
- Physics