{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/154377"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/154377","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Methods to program and to probe RNA tertiary structure with nucleic acid origami","abstract":"Biological structure determination has revolutionized mechanistic understandings, nanotechnology, and drug design. Despite advances in structural determination technologies, from nuclear magnetic resonance to cryo-electron microscopy (cryo-EM), one class of biomolecules has resisted 3D structure characterization. RNA, particularly larger RNAs, often dynamically adopt multiple conformations in a structural ensemble, and this heterogeneity has made 3D structure determination challenging through conventional techniques. In this thesis, I investigated two avenues for improving RNA 3D structure determination, both leveraging the nanoscale programmability of nucleic acid origami. Nucleic acid origami generally involves folding one long single-stranded nucleic acid, the scaffold, into a target geometry via hybridization with short oligonucleotide \"staples.\" First, we expanded the geometric space accessible to 3D wireframe DNA-scaffolded origami with edges composed of two helix bundles, optimizing folding conditions and crossover design and analyzing the final folded 3D structures, for a new design algorithm. I designed a tetrahedral wireframe DNA origami to capture an engineered tRNA via hybridization at three sites. For this complex, I verified stable, cooperative binding, and characterized the 3D structure with cryo-EM, which confirmed binding at all three sites and yielded a 17-Å resolution reconstruction of the tRNA. I also outlined a high-throughput workflow to probe the unknown tertiary structure of a target RNA with varied designs of DNA origami. Additionally, I studied the design of 3D wireframe RNA-scaffolded origami, characterizing the folded structure for several crossover schemes to evaluate how best to accommodate the A-form helical geometry of RNA for robust designs. The resulting algorithm for designing RNA-scaffolded polyhedra enables precise, covalent anchoring of a target RNA fragment onto a wireframe polyhedra. I tested this anchoring approach to attach a 232-nt HIV-1 RNA fragment to an RNA-scaffolded pentagonal bipyramid as a method to improve cryo-EM characterization. The particles folded into the expected pentagonal bipyramidal geometries, and cryo-EM micrographs suggested anchored target RNA, but the design and data analysis need further refinement to determine a 3D structure for the anchored RNA fragment. These studies together represent proofs-of-concept for stabilizing RNA structures on nucleic acid origami, enabled by the expansion of origami design.","abstract_html":"Biological structure determination has revolutionized mechanistic understandings, nanotechnology, and drug design. Despite advances in structural determination technologies, from nuclear magnetic resonance to cryo-electron microscopy (cryo-EM), one class of biomolecules has resisted 3D structure characterization. RNA, particularly larger RNAs, often dynamically adopt multiple conformations in a structural ensemble, and this heterogeneity has made 3D structure determination challenging through conventional techniques. In this thesis, I investigated two avenues for improving RNA 3D structure determination, both leveraging the nanoscale programmability of nucleic acid origami. Nucleic acid origami generally involves folding one long single-stranded nucleic acid, the scaffold, into a target geometry via hybridization with short oligonucleotide "staples." First, we expanded the geometric space accessible to 3D wireframe DNA-scaffolded origami with edges composed of two helix bundles, optimizing folding conditions and crossover design and analyzing the final folded 3D structures, for a new design algorithm. I designed a tetrahedral wireframe DNA origami to capture an engineered tRNA via hybridization at three sites. For this complex, I verified stable, cooperative binding, and characterized the 3D structure with cryo-EM, which confirmed binding at all three sites and yielded a 17-Å resolution reconstruction of the tRNA. I also outlined a high-throughput workflow to probe the unknown tertiary structure of a target RNA with varied designs of DNA origami. Additionally, I studied the design of 3D wireframe RNA-scaffolded origami, characterizing the folded structure for several crossover schemes to evaluate how best to accommodate the A-form helical geometry of RNA for robust designs. The resulting algorithm for designing RNA-scaffolded polyhedra enables precise, covalent anchoring of a target RNA fragment onto a wireframe polyhedra. I tested this anchoring approach to attach a 232-nt HIV-1 RNA fragment to an RNA-scaffolded pentagonal bipyramid as a method to improve cryo-EM characterization. The particles folded into the expected pentagonal bipyramidal geometries, and cryo-EM micrographs suggested anchored target RNA, but the design and data analysis need further refinement to determine a 3D structure for the anchored RNA fragment. These studies together represent proofs-of-concept for stabilizing RNA structures on nucleic acid origami, enabled by the expansion of origami design.","abstract_has_math":false,"creators":["Parsons, Molly F."],"institution":"Massachusetts Institute of Technology","degree_name":"Doctoral","degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Biological Engineering","school":null,"contributors":[],"advisors":["Bathe, Mark"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-05","date_published":"2022-05","updated_at":"2026-07-22T22:22:06Z","subjects":[],"languages":[],"rights":["In Copyright - Educational Use Permitted","Copyright MIT"],"rights_urls":["http://rightsstatements.org/page/InC-EDU/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/1721.1/154377","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Bathe, Mark"]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Biological Engineering"]},{"key":"dc:creator","label":"Author","values":["Parsons, Molly F."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2024-05-01T14:32:04Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2024-05-01T14:32:04Z"]},{"key":"dc:date.issued","label":"Date","values":["2022-05"]},{"key":"dc:publisher","label":"Institution","values":["Massachusetts Institute of Technology"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctoral","Doctor of Philosophy"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright - Educational Use Permitted","Copyright MIT"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/page/InC-EDU/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/1721.1/154377"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Biological structure determination has revolutionized mechanistic understandings, nanotechnology, and drug design. Despite advances in structural determination technologies, from nuclear magnetic resonance to cryo-electron microscopy (cryo-EM), one class of biomolecules has resisted 3D structure characterization. RNA, particularly larger RNAs, often dynamically adopt multiple conformations in a structural ensemble, and this heterogeneity has made 3D structure determination challenging through conventional techniques. In this thesis, I investigated two avenues for improving RNA 3D structure determination, both leveraging the nanoscale programmability of nucleic acid origami. Nucleic acid origami generally involves folding one long single-stranded nucleic acid, the scaffold, into a target geometry via hybridization with short oligonucleotide \"staples.\" First, we expanded the geometric space accessible to 3D wireframe DNA-scaffolded origami with edges composed of two helix bundles, optimizing folding conditions and crossover design and analyzing the final folded 3D structures, for a new design algorithm. I designed a tetrahedral wireframe DNA origami to capture an engineered tRNA via hybridization at three sites. For this complex, I verified stable, cooperative binding, and characterized the 3D structure with cryo-EM, which confirmed binding at all three sites and yielded a 17-Å resolution reconstruction of the tRNA. I also outlined a high-throughput workflow to probe the unknown tertiary structure of a target RNA with varied designs of DNA origami. Additionally, I studied the design of 3D wireframe RNA-scaffolded origami, characterizing the folded structure for several crossover schemes to evaluate how best to accommodate the A-form helical geometry of RNA for robust designs. The resulting algorithm for designing RNA-scaffolded polyhedra enables precise, covalent anchoring of a target RNA fragment onto a wireframe polyhedra. I tested this anchoring approach to attach a 232-nt HIV-1 RNA fragment to an RNA-scaffolded pentagonal bipyramid as a method to improve cryo-EM characterization. The particles folded into the expected pentagonal bipyramidal geometries, and cryo-EM micrographs suggested anchored target RNA, but the design and data analysis need further refinement to determine a 3D structure for the anchored RNA fragment. These studies together represent proofs-of-concept for stabilizing RNA structures on nucleic acid origami, enabled by the expansion of origami design."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Methods to program and to probe RNA tertiary structure with nucleic acid origami"]}]}],"canonical_facts":{"dc:contributor.advisor":["Bathe, Mark"],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Biological Engineering"],"dc:creator":["Parsons, Molly F."],"dc:date.accessioned":["2024-05-01T14:32:04Z"],"dc:date.available":["2024-05-01T14:32:04Z"],"dc:date.issued":["2022-05"],"dc:description.abstract":["Biological structure determination has revolutionized mechanistic understandings, nanotechnology, and drug design. Despite advances in structural determination technologies, from nuclear magnetic resonance to cryo-electron microscopy (cryo-EM), one class of biomolecules has resisted 3D structure characterization. RNA, particularly larger RNAs, often dynamically adopt multiple conformations in a structural ensemble, and this heterogeneity has made 3D structure determination challenging through conventional techniques. In this thesis, I investigated two avenues for improving RNA 3D structure determination, both leveraging the nanoscale programmability of nucleic acid origami. Nucleic acid origami generally involves folding one long single-stranded nucleic acid, the scaffold, into a target geometry via hybridization with short oligonucleotide \"staples.\" First, we expanded the geometric space accessible to 3D wireframe DNA-scaffolded origami with edges composed of two helix bundles, optimizing folding conditions and crossover design and analyzing the final folded 3D structures, for a new design algorithm. I designed a tetrahedral wireframe DNA origami to capture an engineered tRNA via hybridization at three sites. For this complex, I verified stable, cooperative binding, and characterized the 3D structure with cryo-EM, which confirmed binding at all three sites and yielded a 17-Å resolution reconstruction of the tRNA. I also outlined a high-throughput workflow to probe the unknown tertiary structure of a target RNA with varied designs of DNA origami. Additionally, I studied the design of 3D wireframe RNA-scaffolded origami, characterizing the folded structure for several crossover schemes to evaluate how best to accommodate the A-form helical geometry of RNA for robust designs. The resulting algorithm for designing RNA-scaffolded polyhedra enables precise, covalent anchoring of a target RNA fragment onto a wireframe polyhedra. I tested this anchoring approach to attach a 232-nt HIV-1 RNA fragment to an RNA-scaffolded pentagonal bipyramid as a method to improve cryo-EM characterization. The particles folded into the expected pentagonal bipyramidal geometries, and cryo-EM micrographs suggested anchored target RNA, but the design and data analysis need further refinement to determine a 3D structure for the anchored RNA fragment. These studies together represent proofs-of-concept for stabilizing RNA structures on nucleic acid origami, enabled by the expansion of origami design."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["https://hdl.handle.net/1721.1/154377"],"dc:publisher":["Massachusetts Institute of Technology"],"dc:rights":["In Copyright - Educational Use Permitted","Copyright MIT"],"dc:rights.uri":["http://rightsstatements.org/page/InC-EDU/1.0/"],"dc:title":["Methods to program and to probe RNA tertiary structure with nucleic acid origami"],"dc:type":["Thesis"],"thesis:degree_name":["Doctoral","Doctor of Philosophy"]},"updated_at":"2026-07-22T22:22:06Z"}