Ohoka, Ayako2023-04-132023-04-132023-02https://hdl.handle.net/11299/253717University of Minnesota Ph.D. dissertation. 2023. Major: Biomedical Engineering. Advisor: Casim Sarkar. 1 computer file (PDF); x, 185 pages.Biologics, which encompass peptides, proteins, nucleotides, and cell-based products are widely used in both therapeutics and diagnostic applications. Compared to small molecule drugs, biologics can achieve higher efficacy with lower systemic adverse side effects due to their complex three-dimensional structures which allow for better target selectivity and higher affinity. Despite the importance of biologics in human health, their discovery remains highly challenging, costly, and often unfruitful. In the past four decades, there has been an emergence of display technologies, in which a genotype (e.g., RNA) is connected to its corresponding phenotype (e.g., protein) and the library is typically screened or selected for binding against a target of interest. While these display technologies can streamline the discovery process since the amplification and readout of selected proteins can be carried out at the genetic level, current approaches could be further improved to address challenges in the ability to (1) perform selections in complex environments like cell surfaces and within a naïve library and (2) enrich for a more diverse pool of recovered binders, including ones with low-affinity that are typically lost. The molecular engineering approaches and experimental protocols developed in this thesis work improve the signal-to-noise for the specific enrichment of protein binders in an array of biologics discovery platforms. Towards minimizing noise from nonspecific adsorption, we developed PEGylation strategies for stealthing DNA templates for biopanning applications in DNA display. Further, to enrich recovery of true binders and reduce noise from nonfunctional and nonspecific library binders, we introduced methods to incorporate photocrosslinking of the binder library to its target in mRNA display. To improve signal via higher binder recovery of low-affinity binders, we developed a facile cloning method that leverages rolling circle amplification to create homomultivalent protein libraries for in vitro display technologies. Finally, in our collaborative protein work, we made efforts toward engineering biotin ligase fusions to identify new therapeutic targets and understand disease biology with proximity ligation using a blood-brain barrier cell model. Altogether, these advances in molecular engineering contribute to the broader toolbox for the discovery of new biologics.enBiopanningDirected evolutionIn vitro displayThesis or Dissertation