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. In recent years, a new, promising single-molecule biophysical technique called single-molecule field effect transistors (smFETs) provides 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.
My Ph.D. research has spanned the development and establishment of smFETs as a general single-molecule approach for studies of RNA conformational dynamics, and the application of smFETs to understand the underlying molecular mechanism in which RNA stem-loops, a fundamental building block for complex RNA structures, undergo folding and unfolding. In this talk, I will describe the experimental methods that were developed to enable smFET studies of RNA conformational dynamics. This includes a high-throughput fabrication process that generates high signal-to-noise ratio (SNR) smFET devices and nucleic acid tethering strategies that enable controlled tethering of biomolecules onto smFET devices. Utilizing these methods, I first establish smFETs as a general single-molecule approach to characterize conformational dynamics of RNA, then apply smFETs to investigate the molecular mechanism by which a model RNA stem-loop folds and unfolds. These studies provide unique insights into how the organization of the loop structure in the stem-loop shapes its folding energy landscape, and consequently, dictates the kinetic pathway that the stem-loop undergoes during folding.