Harms, MichaelMorrison, AnnelieseHopkins, SamanthaWonderlick, Daria2021-07-272021-07-272021https://hdl.handle.net/1794/2657651 pagesThe ability to engineer biomolecules with new and improved functions could revolutionize medicine, industry, and biotechnology. Efficient bioengineering requires a predictive understanding of the sequence changes needed to produce a desired functional effect. Our work develops a theoretical framework for handling the complications that arise when multiple sequence changes are introduced into a molecular switch. The functional effects of paired mutations can be coupled to each other, and communication between the sites can be mediated by both direct (contact-based) and indirect (ensemble-based) pathways. We designed an experiment to distinguish between these two coupling mechanisms in the adenine riboswitch, a small RNA molecule that switches between different conformations depending on adenine availability. We measured the coupled effects of paired mutations throughout the riboswitch with respect to binding the fluorescent base analog 2-aminopurine. By perturbing the riboswitch’s chemical environment with magnesium ions, we could manipulate the riboswitch’s conformations to isolate direct (within-conformation contacts) and indirect (across-conformation ensemble redistribution) coupling components. While we observed experimental signatures corresponding to both direct and indirect coupling mechanisms, we were unable to successfully tease them apart. This may point to their fundamental interdependence in molecular switches. Efforts to engineer these biomolecules may therefore benefit from predictive models that take both communication pathways into account.en-USCC BY-NC-ND 4.0epistasisensemblesthermodynamicsRNAriboswitchThesis/Dissertation0000-0002-0830-5330