{"id":{"repo_id":"buffalo","oai_identifier":"oai:ubir.buffalo.edu:10477/80687"},"canonical_url":"https://search.dev.ndltd.org/etd/buffalo/oai:ubir.buffalo.edu:10477/80687","repository":{"repo_id":"buffalo","name":"Buffalo","base_url":"https://ubir.buffalo.edu/oai/request"},"display":{"title":"Selection of Ribozyme Kinetic Facilitator","abstract":"M.S.","abstract_html":"M.S.","abstract_has_math":false,"creators":["Jin, Zeng"],"institution":"State University of New York at Buffalo","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Sullivan, John","Pharmacology and Toxicology"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2019,"date_issued":"2019-10-28T22:04:09Z","date_published":"2019-10-28T22:04:09Z","updated_at":"2026-07-27T19:05:23Z","subjects":["pharmacology"],"languages":["eng"],"rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10477/80687","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Sullivan, John","Pharmacology and Toxicology"]},{"key":"dc:creator","label":"Author","values":["Jin, Zeng"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2019-10-28T22:04:09Z","2019","2019-07-31 09:11:32"]},{"key":"dc:publisher","label":"Institution","values":["State University of New York at Buffalo"]},{"key":"dc:type","label":"Dc Type","values":["Text","Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["pharmacology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/10477/80687"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["M.S.","Autosomal dominant retinitis pigmentosa (AdRP) is an eye disorder characterized by the death of photoreceptors leading to a loss of vision. Mutations in the gene for rhodopsin, Rho, is responsible for 30% of all cases. Research has been focused on developing gene therapeutics to knock down the mutated mRNA. Ribozyme is a small catalytic enzyme that is capable of cleaving mRNA substrate. Using ribozyme as a gene therapeutics to knock down Rho mRNA has been halted since it has a relatively slow cleavage rate. Recently, the lab has identified a facilitator sequence that can be added to the 3’ end of ribozyme and dramatically increases the cleavage rate. In order to find a better or other facilitator sequences, we decided to use directed evolution as the approach. The cis evolution demands an effective ribozyme inhibitor during in vitro transcription. We tested neomycin, terbium and cleavage site binding oligonucleotides against our randomized ribozyme and the effects were limited. Therefore, a ribozyme core-binding oligonucleotides were designed and tested to be effective but significantly lowered the RNA products from in vitro transcription. The trans evolution requires labeling of the 5' end of the substrate RNA with a biotin molecule. Traditionally, a minimal 15 nucleotides RNA substrate is purchased pre-labeled with a biotin molecule. In order to mimic the local mRNA structure around the cleavage site, we used a long RNA substrate and labeled it with biotin alternatively by binding to a probe sequence. The results showed an optimal labeling efficiency with minimal noise (3.6%) during the cleavage reaction. Due to the heterogeneity in the PCR sizes after two rounds of evolution, only one trans selection was performed with a cleavage condition of 0.5 mM Mg2+ for 10 minutes, and the noise level was 15.1%. The sequencing results from 60 clones revealed a diversity in the randomized region with a weak GU rich consensus sequence. The structures generated from the consensus sequence are different from each other but mostly a partial stem with loop/budge. Two of the structures were perfect stems, which is the same as the original facilitator but the sequences are different. The effectiveness of the new facilitator sequences remains to be determined. Nevertheless, the diverse nucleotides in the randomized region indicate that there is more than one kinetic facilitator."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Selection of Ribozyme Kinetic Facilitator"]}]}],"canonical_facts":{"dc:contributor":["Sullivan, John","Pharmacology and Toxicology"],"dc:creator":["Jin, Zeng"],"dc:date":["2019-10-28T22:04:09Z","2019","2019-07-31 09:11:32"],"dc:description":["M.S.","Autosomal dominant retinitis pigmentosa (AdRP) is an eye disorder characterized by the death of photoreceptors leading to a loss of vision. Mutations in the gene for rhodopsin, Rho, is responsible for 30% of all cases. Research has been focused on developing gene therapeutics to knock down the mutated mRNA. Ribozyme is a small catalytic enzyme that is capable of cleaving mRNA substrate. Using ribozyme as a gene therapeutics to knock down Rho mRNA has been halted since it has a relatively slow cleavage rate. Recently, the lab has identified a facilitator sequence that can be added to the 3’ end of ribozyme and dramatically increases the cleavage rate. In order to find a better or other facilitator sequences, we decided to use directed evolution as the approach. The cis evolution demands an effective ribozyme inhibitor during in vitro transcription. We tested neomycin, terbium and cleavage site binding oligonucleotides against our randomized ribozyme and the effects were limited. Therefore, a ribozyme core-binding oligonucleotides were designed and tested to be effective but significantly lowered the RNA products from in vitro transcription. The trans evolution requires labeling of the 5' end of the substrate RNA with a biotin molecule. Traditionally, a minimal 15 nucleotides RNA substrate is purchased pre-labeled with a biotin molecule. In order to mimic the local mRNA structure around the cleavage site, we used a long RNA substrate and labeled it with biotin alternatively by binding to a probe sequence. The results showed an optimal labeling efficiency with minimal noise (3.6%) during the cleavage reaction. Due to the heterogeneity in the PCR sizes after two rounds of evolution, only one trans selection was performed with a cleavage condition of 0.5 mM Mg2+ for 10 minutes, and the noise level was 15.1%. The sequencing results from 60 clones revealed a diversity in the randomized region with a weak GU rich consensus sequence. The structures generated from the consensus sequence are different from each other but mostly a partial stem with loop/budge. Two of the structures were perfect stems, which is the same as the original facilitator but the sequences are different. The effectiveness of the new facilitator sequences remains to be determined. Nevertheless, the diverse nucleotides in the randomized region indicate that there is more than one kinetic facilitator."],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/10477/80687"],"dc:language":["eng"],"dc:publisher":["State University of New York at Buffalo"],"dc:rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"dc:subject":["pharmacology"],"dc:title":["Selection of Ribozyme Kinetic Facilitator"],"dc:type":["Text","Thesis"]},"updated_at":"2026-07-27T19:05:23Z"}