It has long been recognized that our understanding of how RNA adapts its complex three-dimensional structure and undergoes conformational fluctuations has played a central role in our understanding of the biological functions of RNA. Our current understanding of the vast and diverse set of RNA conformational dynamics is the culmination of several decades of biophysical research applying several ensemble and single-molecule techniques.
In this journey, each of the biophysical techniques have provided a unique perspective into the dynamic processes of RNA and revealed information about distinct RNA dynamics occurring over a broad range of timescales. In recent years, a new, promising single-molecule biophysical technique called single-molecule field effect transistors (smFETs) has been developed. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that enables single-molecule studies of RNA conformational dynamics observed at microsecond temporal resolution for a long period of time. The broad range of timescales opens immediate prospects for smFETs to provide a unique perspective into understanding RNA conformational dynamics that are presently inaccessible in other single-molecule approaches.
The primary focus of this thesis is to understand how RNA stem-loops undergo folding and unfolding. Stem-loops are one of the most common secondary structural motifs in RNA and act as a fundamental building block for complex RNA structures. Despite their fundamental importance, a complete unifying picture of the folding mechanism of RNA stem-loops has been difficult to achieve, primarily due to the rugged nature of their folding energy landscapes. In Chapter 2, experimental methods that were developed to enable smFET studies of RNA conformational dynamics are described. This includes the development of a high-throughput fabrication process that generates high signal to noise ratio (SNR) smFET devices and the development and validation of nucleic acid tethering strategies that enables controlled tethering of biomolecules onto smFET devices.
Utilizing these methods, Chapter 3 establishes smFET as a general single-molecule approach to characterize the folding dynamics of RNA stem-loops. Finally, Chapter 4 explores the use of smFETs to investigate the molecular mechanism in which a model RNA stem-loop undergoes folding and unfolding. Collectively, this thesis demonstrates how smFETs can be applied to uniquely capture and describe the folding energy landscapes of RNA and reveal new insights to how RNAs fold and unfold.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/fagj-cs27 |
| Date | January 2023 |
| Creators | Jang, Sukjin Steve |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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