eprintid: 8931 rev_number: 5 userid: 6 dir: disk0/00/00/89/31 datestamp: 2011-11-10 19:57:37 lastmod: 2016-11-15 13:48:21 status_changed: 2011-11-10 19:57:37 type: thesis_degree metadata_visibility: show contact_email: sparano@pitt.edu item_issues_count: 0 eprint_status: archive creators_name: Sparano, Brian Andrew creators_email: sparano@pitt.edu creators_id: SPARANO title: The Development of Fluorescent Sensors for Specific RNA Using Chemistry and Combinatorial Biology ispublished: unpub divisions: sch_as_chemistry full_text_status: public keywords: fluorescence spectroscopy; fluorescent sensors; in vitro selection; RNA abstract: Here we describe the development of small molecule-based fluorescent sensors for RNA. The sensors described here and the new paradigm they represent are a convergence of chemical and biological methods. The resulting sensor system consists of two components: a rationally designed small-molecule fluorescent sensor and a reporter RNA sequence that binds to and enhances its fluorescence, isolated with combinatorial biology. First, we rationally designed and synthesized a series of modular fluorescent chemosensors which would be quenched in solution and fluorescent only when bound to their target. These sensors consist of a fluorophore (2',7'-dichlorofluorescein, DCF) conjugated to a pair of quencher units (aniline derivatives in this case) which quench the fluorescence of DCF by the PET (photoinduced electron transfer) mechanism. NMR and fluorescent spectroscopic analyses of these DCF derivatives revealed important correlations between their conformations, the PET, and fluorescent intensity in addition to insight on their solvatochromism and pH dependence. These studies allowed us to choose a candidate sensor for the next step: using combinatorial biology to isolate a reporter sequence. In order to isolate RNA that would enhance the fluorescence of our sensor, RNA that bound to the aniline-based quencher was isolated via in vitro selection (a.k.a SELEX or systematic evolution of ligands by exponential enrichment). These RNA sequences (aptamers) were then screened for their ability to switch on the fluorescence of the sensor. One of these aptamers was found to enhance the fluorescence intensity of the DCF-aniline conjugate in a concentration-dependent manner, leading to further study of that aptamer with mutation studies and additional in vitro selection experiments. To demonstrate the power and generality of this approach, a parallel in vitro selection was performed with aptamers from this selection having similar activities. These results show that one can develop fluorescence-inducing reporter RNA and morph it into remotely related sequences without prior structural insight into RNA-ligand binding through the rational design of fluorescent chemosensors and subsequent isolation of RNA from combinatorial libraries. date: 2007-09-27 date_type: completed institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_name: Koide, Kazunori etdcommittee_name: Day, Billy W etdcommittee_name: Schafmeister, Chris etdcommittee_name: Weber, Stephen etdcommittee_email: koide@pitt.edu etdcommittee_email: bday@pitt.edu etdcommittee_email: sweber@pitt.edu etdcommittee_id: KOIDE etdcommittee_id: BDAY etdcommittee_id: SWEBER etd_defense_date: 2007-08-09 etd_approval_date: 2007-09-27 etd_submission_date: 2007-08-06 etd_access_restriction: 5_year etd_patent_pending: FALSE assigned_doi: doi:10.5195/pitt.etd.2011.8931 thesis_type: dissertation degree: PhD committee: Kazunori Koide (koide@pitt.edu) - Committee Chair committee: Billy W. Day (bday@pitt.edu) - Committee Member committee: Chris Schafmeister () - Committee Member committee: Stephen Weber (sweber@pitt.edu) - Committee Member etdurn: etd-08062007-141424 other_id: http://etd.library.pitt.edu/ETD/available/etd-08062007-141424/ other_id: etd-08062007-141424 citation: Sparano, Brian Andrew (2007) The Development of Fluorescent Sensors for Specific RNA Using Chemistry and Combinatorial Biology. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/8931/1/Sparano_8-13-07.pdf