The conformational dynamics of RNA are crucial to its role in numerous essential biological functions, requiring a comprehensive view of masses of individual molecular motions in order to fully understand these processes. Obtaining such a unified view, however, presents many challenges. Even the simplest RNA structures undergo rearrangements within an intricate three-dimensional network of secondary and tertiary interactions, resulting in motions that span a broad range of timescales.
This complexity gives rise to a large number of experimental techniques sampling different aspects of the folding process, leaving a rather fragmented picture of RNA folding overall. In order to address the divergence and limitations of existing ensemble and single-molecule methods, this work describes the application of carbon nanotube (CNT)-based single-molecule field-effect transistors (smFET) as a platform for studying the folding and unfolding dynamics of RNA. smFET is capable of measuring individual biomolecular dynamics for long durations, while at a sufficiently high time resolution to capture the relevant timescales for RNA folding.
This technique, moreover, avoids potential sources of interference or damage to the molecule as found in other available methods, and capitalizes on the detailed information that can be garnered from studying a single molecule as opposed to an ensemble. By taking advantage of the highly sensitive electronic properties and nanoscale dimensions of CNTs, and tethering a comparably sized and charged single biomolecule like RNA, it is possible to monitor the folding and unfolding of the molecule and characterize the kinetics associated with these motions. This thesis describes the optimization and application of such smFET technology to RNA stem-loops, which are extremely prevalent and thermodynamically stable elements of RNA secondary structure.
Chapter 1 introduces the biological context of these molecules as well as the mechanisms of CNT-based smFET sensing. In Chapter 2, the methods used to fabricate smFET devices are described along with the experiments conducted to optimize single-molecule tethering. These methods and protocols were then applied to the studies detailed in Chapter 3, which examines the implications of the thermodynamic and kinetic analyses of RNA stem-loop folding and unfolding as investigated with smFET. Chapter 4 concludes with a brief overview of what has been accomplished and potential future directions for this platform.
The work expressed here thus presents a cohesive view of RNA stem-loop folding, integrating the past results of both experimental and computational studies. With this improved smFET methodology, this technique can be applied to many other essential and increasingly complex biological systems to achieve a fuller and richer understanding of the processes that govern life.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/dqrh-wc42 |
| Date | January 2022 |
| Creators | Dubnik, Sarah |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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