This thesis focuses on three separate explorations into the nature of toehold-mediated strand displacement which is a reaction characterized by the swapping of one stably bound oligonucleotide for another. The first study seeks to improve current methodologies in single-molecule surface experiments using strand displacement. Presently, surfaces are prepared through time-consuming and costly cleaning and functionalization protocols which are only useful for a single trial of an experiment due to the irreversible binding of target molecules to the surface. To solve this problem, we develop a new method called ERASE which is powered by the inherent switching nature of strand displacement. Further, we showcase the wide applicability and adaptability of ERASE to different biophysical systems of interest. Next, we use single-molecule fluorescence to study the effect of single mismatch position on strand displacement kinetics. We find a significant increase in the mean first passage time with mismatches proximal to the toehold. We highlight the 1D model's failure to account for the observed position dependence. Further, we show that addition of direct dissociation to the 1D branch migration model explains the observed data. To provide biophysical insight, we propose a simpler three-step model that explains mismatch-dependent behavior. Finally, we explore the biophysics of the branch migration subprocess. We design FRET constructs to decouple the short-lived branch migration process from the overwhelmingly dominant toehold binding step. We further show a wide sequence-dependent diversity of waiting time distributions. We measure distribution changes between systems of "complementary invasion" which hint to dangling ends as the origin. Moreover, we design comparatively lower-cost single fluorophore constructs which allow for large scale sequence and length dependent studies.
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Single-molecule biophysics of toehold-mediated strand displacement
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