Tompkins, Kassidy2021-10-252021-10-252021-01https://hdl.handle.net/11299/225129University of Minnesota Ph.D. dissertation.January 2020. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Wendy Gordon. 1 computer file (PDF); viii, 126 pages.Bioconjugation is a broad technique used to form direct linkages between and biomolecule and another functional chemical. More specifically, coupling functional proteins to programmable nucleic acids has enabled biotechnologies ranging from therapeutic diagnostics to super-resolution microscopy, indispensable for medical professionals and researchers alike. While current protein-nucleic acid bioconjugation methods have sprouted a myriad of biotechnologies, only a small number of compatible orthogonal, or non-cross-reactive, chemistries exist posing a bottleneck for multiplexing. In recent years, our research group has exploited HUH-endonucleases as fusion tags, dubbed HUH-tags, to create covalent adducts with specific single-stranded DNA (ssDNA) sequences. This dissertation addresses foundational biochemical knowledge and protein engineering vital for development of multiplexed HUH-tag applications. The ultimate goal of this research is to develop a toolkit of multiplexable HUH-tags ready for implementation into a wide variety of platforms. As a starting point, we explored the basis of ssDNA recognition of replication initiator proteins (Reps), which are a highly versatile version of HUH-tag that process DNA faster and are more compact than other HUH-endonucleases. Through analysis of co-crystal structures and development of a deep sequencing-based cleavage assay, HUH-seq, we demonstrate how Reps with subtle differences in specificity can be used as orthogonal HUH-tags and how specificity can be predictably engineered. Next, we characterized the cleavage kinetics of Reps and show how Reps from different families of organisms can reach completion in seconds. Finally, we show an example of rational Rep engineering by circular permutation, and how Reps can be exploited to form protein-RNA conjugates. The characterization and protein engineering described in this dissertation demonstrate how HUH-tags are a critical emerging bioconjugation technology.enbioconjugationHUH-endonucleasenucleic acid binding proteinprotein engineeringreplication initiator proteinsingle-stranded DNA nucleaseThesis or Dissertation