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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorMcGown, Linda Baine
dc.contributorLigon, Lee
dc.contributorLakshmi, K. V.
dc.contributorLinhardt, Robert J.
dc.contributor.authorZhang, Tian
dc.date.accessioned2021-11-03T08:08:25Z
dc.date.available2021-11-03T08:08:25Z
dc.date.created2014-04-14T11:29:46Z
dc.date.issued2013-12
dc.identifier.urihttps://hdl.handle.net/20.500.13015/1068
dc.descriptionDecember 2013
dc.descriptionSchool of Science
dc.description.abstractThe second part of this dissertation focuses on the fundamental characterization of affinity surfaces. Specifically, we studied affinity protein capture at G-quadruplex-forming-aptamer-modified surfaces using imaging tools. Two systems were used to investigate the DNA-modification and protein capture characteristics of different affinity surfaces: first, thrombin binding aptamer (TBA) and a control oligo sequence with no G-quadruplex forming ability, and second, the Pu28-mer sequence. The affinity surfaces of interest included fused silica plates, streptavidinated magnetic beads, and indium tin oxide (ITO)-coated glass slides. The results of these experiments provide a better fundamental understanding of protein capture at aptamer-modified surfaces that might lead to improved design and reliability of devices and processes for protein analysis, separation, isolation and purification.
dc.description.abstractThe first part of the dissertation focuses on in vitro investigation of G4 formation by Pu28-mer, a 28bp GGA-TCC repeat sequence in the promoter region of the gene ERBB2 that expresses the protein ErbB2, and identification of its binding proteins both in vitro and in live cells. Human epidermal growth factor receptor 2 (ErbB2, or Her2/neu) is overexpressed in as much as 30% of human breast cancers, making it a key breast cancer marker protein. It has been suggested that the regulatory mechanism of the ERBB2 promoter region may involve formation of a G-quadruplex (G4) structure, possibly similar to the tetrad:heptad:heptad:tetrad (T:H:H:T) G4 structure formed by the analogous, GGA-rich sequence from the promoter region of the c-MYB proto-oncogene. In order to investigate this possibility, circular dichroism (CD) spectroscopy of the Pu28-mer and in vitro protein capture from MCF-7 cell nuclear extracts at Pu28-mer and control oligonucleotide-modified surfaces were performed. Captured proteins were analyzed by LC-MS/MS and further interrogated using Western blotting and chromatin immunoprecipitation (ChIP). Results support the formation of T:H:H:T structure and indicate that Pu28-mer selectively captures transcription factors including Ku70, Ku80, and PURA. The results may add to the understanding of the role of non-duplex DNA structures in gene regulation and provide a more complete picture of the regulation of ErbB2 expression in breast cancer. The results also provide a blueprint for development of "genome-inspired" aptamers based on the Pu28-mer sequence for detection of proteins related to regulation of ErbB2 expression and breast cancer.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectChemistry
dc.titleProtein capture and identification at G-quadruplex DNA affinity surfaces
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid170956
dc.digitool.pid170957
dc.digitool.pid170958
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentDept. of Chemistry and Chemical Biology


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