{"id":{"repo_id":"penn","oai_identifier":"oai:repository.upenn.edu:20.500.14332/30207"},"canonical_url":"https://search.dev.ndltd.org/etd/penn/oai:repository.upenn.edu:20.500.14332/30207","repository":{"repo_id":"penn","name":"University of Pennsylvania","base_url":"https://repository.upenn.edu/server/oai/request"},"display":{"title":"Next-Generation Oligonucleotide Probes For Transcriptome In Vivo Analysis (tiva)","abstract":"The transcriptome ‒ the total collection of every RNA transcript in a cell ‒ provides a unique readout of a cell’s commands as it executes its own genetic code. Studying the transcriptomes of individual cells is a powerful way to identify new cell types, building a better understanding of complex tissues from the ground up. Furthermore, transcriptomic characterization of tumor samples is already leading to tangible advancements in personalized cancer care. However, the process of capturing a transcriptome requires careful isolation of mRNA from a single cell that is potentially entrenched in an entangled, three-dimensional tissue structure. Our lab has previously developed a method to successfully isolate mRNA from single cells still contextualized within living tissue. This method, Transcriptome In Vivo Analysis (TIVA), utilizes a light-activatable oligonucleotide probe to offer fine spatio-temporal control of mRNA capture. TIVA probes are highly modified RNA hairpins or loops incorporating a poly(U) “capture” sequence complementary to the poly(A) tail of mRNA, as well as a biotin moiety to enable pull-down of bound mRNA. To enable spatio-temporal control of mRNA binding, the probes are locked into an inactive “caged” conformation. Laser excitation of the target cell severs photoactivable o-nitrobenzyl- or Ru(II) polypyridyl-based linkages built into the probe, freeing the poly(U) capture sequence to effectively biotinylate mRNA. After photolysis, a small tissue region containing the target cell is aspirated out and lysed so that the target mRNA can be isolated by streptavidin-biotin affinity purification. In this dissertation I present the synthesis, characterization, and application of next-generation TIVA constructs that aim to address various limitations of the original probe. (I) I demonstrate that phosphorothioation extends the serum stability of the TIVA probe from a few minutes to over 24 hours, and mediates uptake into cells without the need for a cell-penetrating peptide. (II) I also present versions of the probe with hairpin-terminating GC pairs, longer blocking strands, and a pegylated hairpin turn which dramatically reduce the probe’s pre-photolysis background binding of mRNA. (III) Finally, I show that incorporation of a Ru(II) polypyridyl-based photocleavable linker extends the probe’s activation response from 1-photon near-UV light to 2-photon near-IR light. Together, these advancements move TIVA towards a broader range of applications – including deeper regions of challenging, nuclease-abundant tissues – with greater confidence that our construct will remain stable and generate a low-background transcriptome.","abstract_html":"The transcriptome ‒ the total collection of every RNA transcript in a cell ‒ provides a unique readout of a cell’s commands as it executes its own genetic code. Studying the transcriptomes of individual cells is a powerful way to identify new cell types, building a better understanding of complex tissues from the ground up. Furthermore, transcriptomic characterization of tumor samples is already leading to tangible advancements in personalized cancer care. However, the process of capturing a transcriptome requires careful isolation of mRNA from a single cell that is potentially entrenched in an entangled, three-dimensional tissue structure. Our lab has previously developed a method to successfully isolate mRNA from single cells still contextualized within living tissue. This method, Transcriptome In Vivo Analysis (TIVA), utilizes a light-activatable oligonucleotide probe to offer fine spatio-temporal control of mRNA capture. TIVA probes are highly modified RNA hairpins or loops incorporating a poly(U) “capture” sequence complementary to the poly(A) tail of mRNA, as well as a biotin moiety to enable pull-down of bound mRNA. To enable spatio-temporal control of mRNA binding, the probes are locked into an inactive “caged” conformation. Laser excitation of the target cell severs photoactivable o-nitrobenzyl- or Ru(II) polypyridyl-based linkages built into the probe, freeing the poly(U) capture sequence to effectively biotinylate mRNA. After photolysis, a small tissue region containing the target cell is aspirated out and lysed so that the target mRNA can be isolated by streptavidin-biotin affinity purification. In this dissertation I present the synthesis, characterization, and application of next-generation TIVA constructs that aim to address various limitations of the original probe. (I) I demonstrate that phosphorothioation extends the serum stability of the TIVA probe from a few minutes to over 24 hours, and mediates uptake into cells without the need for a cell-penetrating peptide. (II) I also present versions of the probe with hairpin-terminating GC pairs, longer blocking strands, and a pegylated hairpin turn which dramatically reduce the probe’s pre-photolysis background binding of mRNA. (III) Finally, I show that incorporation of a Ru(II) polypyridyl-based photocleavable linker extends the probe’s activation response from 1-photon near-UV light to 2-photon near-IR light. Together, these advancements move TIVA towards a broader range of applications – including deeper regions of challenging, nuclease-abundant tissues – with greater confidence that our construct will remain stable and generate a low-background transcriptome.","abstract_has_math":false,"creators":["Yeldell, Sean Brian"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ivan J. Dmochowski"],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018","date_published":"2018","updated_at":"2026-07-24T03:45:49Z","subjects":[],"languages":["en"],"rights":["Sean Brian Yeldell"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://repository.upenn.edu/handle/20.500.14332/30207","outbound_label":"Repository record","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ivan J. Dmochowski"]},{"key":"dc:creator","label":"Author","values":["Yeldell, Sean Brian"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2023-05-17T22:02:06.000"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2023-05-22T17:34:57Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2021-12-21T00:00:00Z"]},{"key":"dc:date.issued","label":"Date","values":["2018"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation/Thesis"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Sean Brian Yeldell"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://repository.upenn.edu/handle/20.500.14332/30207"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The transcriptome ‒ the total collection of every RNA transcript in a cell ‒ provides a unique readout of a cell’s commands as it executes its own genetic code. Studying the transcriptomes of individual cells is a powerful way to identify new cell types, building a better understanding of complex tissues from the ground up. Furthermore, transcriptomic characterization of tumor samples is already leading to tangible advancements in personalized cancer care. However, the process of capturing a transcriptome requires careful isolation of mRNA from a single cell that is potentially entrenched in an entangled, three-dimensional tissue structure. Our lab has previously developed a method to successfully isolate mRNA from single cells still contextualized within living tissue. This method, Transcriptome In Vivo Analysis (TIVA), utilizes a light-activatable oligonucleotide probe to offer fine spatio-temporal control of mRNA capture. TIVA probes are highly modified RNA hairpins or loops incorporating a poly(U) “capture” sequence complementary to the poly(A) tail of mRNA, as well as a biotin moiety to enable pull-down of bound mRNA. To enable spatio-temporal control of mRNA binding, the probes are locked into an inactive “caged” conformation. Laser excitation of the target cell severs photoactivable o-nitrobenzyl- or Ru(II) polypyridyl-based linkages built into the probe, freeing the poly(U) capture sequence to effectively biotinylate mRNA. After photolysis, a small tissue region containing the target cell is aspirated out and lysed so that the target mRNA can be isolated by streptavidin-biotin affinity purification. In this dissertation I present the synthesis, characterization, and application of next-generation TIVA constructs that aim to address various limitations of the original probe. (I) I demonstrate that phosphorothioation extends the serum stability of the TIVA probe from a few minutes to over 24 hours, and mediates uptake into cells without the need for a cell-penetrating peptide. (II) I also present versions of the probe with hairpin-terminating GC pairs, longer blocking strands, and a pegylated hairpin turn which dramatically reduce the probe’s pre-photolysis background binding of mRNA. (III) Finally, I show that incorporation of a Ru(II) polypyridyl-based photocleavable linker extends the probe’s activation response from 1-photon near-UV light to 2-photon near-IR light. Together, these advancements move TIVA towards a broader range of applications – including deeper regions of challenging, nuclease-abundant tissues – with greater confidence that our construct will remain stable and generate a low-background transcriptome."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy (PhD)"]},{"key":"dc:format.mimetype","label":"Dc Format Mimetype","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Next-Generation Oligonucleotide Probes For Transcriptome In Vivo Analysis (tiva)"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ivan J. Dmochowski"],"dc:creator":["Yeldell, Sean Brian"],"dc:date":["2023-05-17T22:02:06.000"],"dc:date.accessioned":["2023-05-22T17:34:57Z"],"dc:date.available":["2021-12-21T00:00:00Z"],"dc:date.issued":["2018"],"dc:description.abstract":["The transcriptome ‒ the total collection of every RNA transcript in a cell ‒ provides a unique readout of a cell’s commands as it executes its own genetic code. Studying the transcriptomes of individual cells is a powerful way to identify new cell types, building a better understanding of complex tissues from the ground up. Furthermore, transcriptomic characterization of tumor samples is already leading to tangible advancements in personalized cancer care. However, the process of capturing a transcriptome requires careful isolation of mRNA from a single cell that is potentially entrenched in an entangled, three-dimensional tissue structure. Our lab has previously developed a method to successfully isolate mRNA from single cells still contextualized within living tissue. This method, Transcriptome In Vivo Analysis (TIVA), utilizes a light-activatable oligonucleotide probe to offer fine spatio-temporal control of mRNA capture. TIVA probes are highly modified RNA hairpins or loops incorporating a poly(U) “capture” sequence complementary to the poly(A) tail of mRNA, as well as a biotin moiety to enable pull-down of bound mRNA. To enable spatio-temporal control of mRNA binding, the probes are locked into an inactive “caged” conformation. Laser excitation of the target cell severs photoactivable o-nitrobenzyl- or Ru(II) polypyridyl-based linkages built into the probe, freeing the poly(U) capture sequence to effectively biotinylate mRNA. After photolysis, a small tissue region containing the target cell is aspirated out and lysed so that the target mRNA can be isolated by streptavidin-biotin affinity purification. In this dissertation I present the synthesis, characterization, and application of next-generation TIVA constructs that aim to address various limitations of the original probe. (I) I demonstrate that phosphorothioation extends the serum stability of the TIVA probe from a few minutes to over 24 hours, and mediates uptake into cells without the need for a cell-penetrating peptide. (II) I also present versions of the probe with hairpin-terminating GC pairs, longer blocking strands, and a pegylated hairpin turn which dramatically reduce the probe’s pre-photolysis background binding of mRNA. (III) Finally, I show that incorporation of a Ru(II) polypyridyl-based photocleavable linker extends the probe’s activation response from 1-photon near-UV light to 2-photon near-IR light. Together, these advancements move TIVA towards a broader range of applications – including deeper regions of challenging, nuclease-abundant tissues – with greater confidence that our construct will remain stable and generate a low-background transcriptome."],"dc:description.degree":["Doctor of Philosophy (PhD)"],"dc:format.mimetype":["application/pdf"],"dc:identifier.uri":["https://repository.upenn.edu/handle/20.500.14332/30207"],"dc:language":["en"],"dc:rights":["Sean Brian Yeldell"],"dc:title":["Next-Generation Oligonucleotide Probes For Transcriptome In Vivo Analysis (tiva)"],"dc:type":["Dissertation/Thesis"]},"updated_at":"2026-07-24T03:45:49Z"}