Limon, Lidia2025-01-282025-01-282023-08https://hdl.handle.net/11299/269643University of Minnesota Ph.D. dissertation. August 2023. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Wendy Gordon. 1 computer file (PDF); viii, 86 pages.Bioconjugation of protein to DNA is increasingly used to enablebiotechnology applications. The ability to link proteins to DNA has become an integral part of scientific research that allows researchers to extract information from proteins and protein complexes using high-throughput methods. While the current bioconjugation methods have served their purpose and continue to be applied by many, the chemistry required poses a number of limitations: specifically, the lack of reactive groups in proteins and DNA. To circumvent this, we and others have exploited HUH-endonucleases to covalently link proteins to DNA. The ability of the Rep domain of HUH-endonucleases to recognize a short nonanucleotide DNA sequence has provided a way to avoid the chemical modifications of proteins and DNA. When used as fusion proteins that we call HUH-tags, the Rep domain can be implemented as self-labeling enzymes for applications reliant on protein-DNA bioconjugation. The objective of this research is to continue to expand and demonstrate the utility of HUH-tags with mammalian proteins in mammalian cells. As a first step to achieving this goal, we explore the natural diversity of the HUH superfamily of endonucleases for Reps. We identify the potential Reps that are compatible with mammalian expression of complex proteins. Through the characterization of Reps in mammalian cells, we develop and validate the functionality of HUH-tags for the production of recombinant antibodies. Finally, we demonstrate the application of HUH-tagged antibodies in DNA-based tension probes and cellular barcoding. The promise of HUH-tags as a bioconjugation method offers an exciting opportunity for novel biotechnology applications.enBioconjugationHUH-endonucleasesThesis or Dissertation