{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/125764"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/125764","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Methods toward investigating viral RNA capping enzymes and antibody breadth expansion","abstract":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","abstract_html":"Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","abstract_has_math":false,"creators":["Ornelas, Marya Ysabelle"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Chemistry","degree_department":null,"school":null,"contributors":["Wu, Nicholas C","Mehta, Angad P","Hergenrother, Paul J","Mitchell, Douglas A","Brooke, Chris C"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-07-07","date_published":"2024-07-07","updated_at":"2026-07-22T22:25:02Z","subjects":["Viral Rna Capping Enzymes","Antibody Breadth Expansion"],"languages":["en","eng"],"rights":["Copyright 2024 Marya Ornelas"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2142/125764","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Wu, Nicholas C","Mehta, Angad P","Hergenrother, Paul J","Mitchell, Douglas A","Brooke, Chris C"]},{"key":"dc:creator","label":"Author","values":["Ornelas, Marya Ysabelle"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2024-07-07","2024-08"]},{"key":"dc:type","label":"Dc Type","values":["text","Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Chemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Viral Rna Capping Enzymes","Antibody Breadth Expansion"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en","eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2024 Marya Ornelas"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://hdl.handle.net/2142/125764"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","The student, Marya Ornelas, accepted the attached license on 2024-06-27 at 12:21.","The student, Marya Ornelas, submitted this Dissertation for approval on 2024-06-27 at 12:41.","This Dissertation was approved for publication on 2024-07-07 at 16:46.","DSpace SAF Submission Ingestion Package generated from Vireo submission #20886 on 2025-02-04 at 21:25:08","With the continued emergence of highly pathogenic RNA and DNA viruses, there is a need to develop modular therapeutic discovery platforms that can be quickly adapted to the emerging virus. Moreover, there is a need for broadly protective vaccines to protect against current and future viral outbreaks. This dissertation encompasses two main projects toward these goals. The first project (Chapters 1 and 2) describes the development of yeast platforms to characterize, evolve, and target viral RNA capping enzymes from RNA and DNA viruses. The second project (Chapter 3) demonstrates the use of a library-on-library yeast display screening approach to analyze the evolution of antibody breadth. The first and second chapters of this dissertation are reprints of papers published in ACS Synthetic Biology and ACS Chemical Biology, respectively. In these studies, we aimed to develop yeast platforms to characterize viral RNA capping enzyme activity in vivo in order to address the three main limitations of previous platforms: 1) the lack of robust in vitro characterization systems, 2) the lack of in vivo eukaryotic systems that provide more accurate biological environments for studying viral protein function and 3) the lack of broadly applicable platforms that can be rapidly adapted to characterize and screen enzymes from emerging pathogenic viruses. We were able to perform biochemical characterization of the N7-methyltransferases (N7-MTases) from SARS-CoV-2, Monkeypox virus (MPV), and African Swine fever virus (ASFV). Further, we identified attenuated variants of these N7-MTases using directed evolution for their use in live attenuated vaccines. We also demonstrated that these yeast strains can be used in high throughput medicinal chemistry applications. Overall, these studies provide modular phenotypic screening platforms to the broader scientific community and may accelerate the development of broad spectrum antivirals and live attenuated vaccine candidates. The third chapter of this dissertation is a reprint of an article submitted for publication (bioRxiv DOI: https://doi.org/10.1101/2024.06.06.597810). Studying the evolution of antibody breadth has been a long-standing challenge in antibody research due to a lack of high-throughput experimental approaches to characterize antibody affinity maturation against multiple antigenic variants in parallel. We addressed this challenge by applying a high-throughput library-on-library approach to determine the binding affinity landscapes of a broadly neutralizing betacoronavirus antibody, S2P6, against 27 unique stem helix peptides across all betacoronavirus subgenera in a single yeast display experiment. This study provides a proof-of-concept for using a yeast display library-on-library approach to analyze the evolution of antibody cross-reactivity breadth and elucidates molecular insights into the development of broadly protective coronavirus vaccines."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Methods toward investigating viral RNA capping enzymes and antibody breadth expansion"]}]}],"canonical_facts":{"dc:contributor":["Wu, Nicholas C","Mehta, Angad P","Hergenrother, Paul J","Mitchell, Douglas A","Brooke, Chris C"],"dc:creator":["Ornelas, Marya Ysabelle"],"dc:date":["2024-07-07","2024-08"],"dc:description":["Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2026-08-01","The student, Marya Ornelas, accepted the attached license on 2024-06-27 at 12:21.","The student, Marya Ornelas, submitted this Dissertation for approval on 2024-06-27 at 12:41.","This Dissertation was approved for publication on 2024-07-07 at 16:46.","DSpace SAF Submission Ingestion Package generated from Vireo submission #20886 on 2025-02-04 at 21:25:08","With the continued emergence of highly pathogenic RNA and DNA viruses, there is a need to develop modular therapeutic discovery platforms that can be quickly adapted to the emerging virus. Moreover, there is a need for broadly protective vaccines to protect against current and future viral outbreaks. This dissertation encompasses two main projects toward these goals. The first project (Chapters 1 and 2) describes the development of yeast platforms to characterize, evolve, and target viral RNA capping enzymes from RNA and DNA viruses. The second project (Chapter 3) demonstrates the use of a library-on-library yeast display screening approach to analyze the evolution of antibody breadth. The first and second chapters of this dissertation are reprints of papers published in ACS Synthetic Biology and ACS Chemical Biology, respectively. In these studies, we aimed to develop yeast platforms to characterize viral RNA capping enzyme activity in vivo in order to address the three main limitations of previous platforms: 1) the lack of robust in vitro characterization systems, 2) the lack of in vivo eukaryotic systems that provide more accurate biological environments for studying viral protein function and 3) the lack of broadly applicable platforms that can be rapidly adapted to characterize and screen enzymes from emerging pathogenic viruses. We were able to perform biochemical characterization of the N7-methyltransferases (N7-MTases) from SARS-CoV-2, Monkeypox virus (MPV), and African Swine fever virus (ASFV). Further, we identified attenuated variants of these N7-MTases using directed evolution for their use in live attenuated vaccines. We also demonstrated that these yeast strains can be used in high throughput medicinal chemistry applications. Overall, these studies provide modular phenotypic screening platforms to the broader scientific community and may accelerate the development of broad spectrum antivirals and live attenuated vaccine candidates. The third chapter of this dissertation is a reprint of an article submitted for publication (bioRxiv DOI: https://doi.org/10.1101/2024.06.06.597810). Studying the evolution of antibody breadth has been a long-standing challenge in antibody research due to a lack of high-throughput experimental approaches to characterize antibody affinity maturation against multiple antigenic variants in parallel. We addressed this challenge by applying a high-throughput library-on-library approach to determine the binding affinity landscapes of a broadly neutralizing betacoronavirus antibody, S2P6, against 27 unique stem helix peptides across all betacoronavirus subgenera in a single yeast display experiment. This study provides a proof-of-concept for using a yeast display library-on-library approach to analyze the evolution of antibody cross-reactivity breadth and elucidates molecular insights into the development of broadly protective coronavirus vaccines."],"dc:format":["application/pdf"],"dc:identifier":["https://hdl.handle.net/2142/125764"],"dc:language":["en","eng"],"dc:rights":["Copyright 2024 Marya Ornelas"],"dc:subject":["Viral Rna Capping Enzymes","Antibody Breadth Expansion"],"dc:title":["Methods toward investigating viral RNA capping enzymes and antibody breadth expansion"],"dc:type":["text","Thesis"],"thesis:degree_discipline":["Chemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:25:02Z"}