{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/113972"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/113972","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Spatially-resolved transcriptomic mapping in live cells using peroxidase-mediated proximity biotinylation","abstract":"The spatial organization of RNA within cells is crucial for the regulation of a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we developed two methods, termed APEX-RIP and APEX-Seq. APEX-RIP combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking, while APEX-Seq utilizes peroxidase-catalyzed in situ biotinylation on RNA. We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts.","abstract_html":"The spatial organization of RNA within cells is crucial for the regulation of a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we developed two methods, termed APEX-RIP and APEX-Seq. APEX-RIP combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking, while APEX-Seq utilizes peroxidase-catalyzed in situ biotinylation on RNA. We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts.","abstract_has_math":false,"creators":["Kaewsapsak, Pornchai"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Chemistry.","school":null,"contributors":[],"advisors":["Alice Y. Ting."],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017","date_published":"2017","updated_at":"2026-07-22T22:22:01Z","subjects":["Chemistry."],"languages":["eng"],"rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/113972","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Alice Y. Ting."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Chemistry."]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. 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They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/113972"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.","Cataloged from PDF version of thesis.","Includes bibliographical references."]},{"key":"dc:description.abstract","label":"Abstract","values":["The spatial organization of RNA within cells is crucial for the regulation of a wide range of biological functions, spanning all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we developed two methods, termed APEX-RIP and APEX-Seq. APEX-RIP combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking, while APEX-Seq utilizes peroxidase-catalyzed in situ biotinylation on RNA. We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:title","label":"Title","values":["Spatially-resolved transcriptomic mapping in live cells using peroxidase-mediated proximity biotinylation"]}]}],"canonical_facts":{"dc:contributor.advisor":["Alice Y. Ting."],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Chemistry."],"dc:contributor.other":["Massachusetts Institute of Technology. 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We demonstrated that APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, bulk cytosol, and endoplasmic reticulum (ER) membrane, with higher specificity and coverage than conventional approaches. We furthermore identified candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Similarly, APEX-Seq can isolate RNAs from mitochondrial matrix, ER-associated RNAs, OMM-associated RNAs, and potentially other non-membrane bound compartments. We also revealed many non-coding RNA candidates at these sites. Since APEX-RIP and APEX-Seq are simple, versatile, and do not require special instrumentation, we envision their broad applications in a variety of biological contexts."],"dc:description.degree":["Ph. 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