Thermodynamic Analysis of Aptamer-Ligand Binding by Isothermal Titration Calorimetry
| dc.contributor.advisor | Johnson, Philip E. | |
| dc.contributor.author | Slavkovic, Sladjana | |
| dc.date.accessioned | 2020-08-11T12:47:28Z | |
| dc.date.available | 2020-08-11T12:47:28Z | |
| dc.date.copyright | 2020-04 | |
| dc.date.issued | 2020-08-11 | |
| dc.date.updated | 2020-08-11T12:47:28Z | |
| dc.degree.discipline | Chemistry | |
| dc.degree.level | Doctoral | |
| dc.degree.name | PhD - Doctor of Philosophy | |
| dc.description.abstract | Aptamers are short, single stranded nucleic acid molecules typically 15 to 60 nucleotides in length with the capacity of binding diverse molecular targets, ranging from small molecules to whole cells primarily due to their specific three-dimensional structure. The cocaine binding aptamer is a DNA aptamer that contains 3 stems built around a 3-way junction. This aptamer was selected by Stojanovic in 2000 using classic SELEX to bind cocaine, but not for its common metabolites, benzoylecgonine and ecgonine methyl ester. It is widely used as a model system in the development of a variety of biosensor applications. The aim of this research was to gain an insight into understanding how aptamers interact with their ligands by measuring thermodynamics. Since very little work has been done in this field using isothermal titration calorimetry (ITC) studies, the thermodynamics of small molecule binding to the cocaine-binding aptamer was investigated in detail. The study included both quinine-based and non-quinine based antimalarial compounds. Some of the results that this study yielded are the importance of a quinoline ring in the ligand, the second binding site on the aptamer, the new tightest binding ligand for the cocaine binding aptamer amodiaquine, and a ligand (artemisinin) that does not contain quinoline ring but binds tightly to the cocaine-binding aptamer. In order to determine the selectivity of the antimalarial compounds (amodiaquine, mefloquine, chloroquine and quinine) for the cocaine-binding aptamer, the investigation was further expanded to other DNA structures such as three-way junctions and duplex DNA of varying length. Results showed that quinine and chloroquine are specific for the cocaine binding aptamer, while amodiaquine binds DNA in general. Artemisinin, a non-quinine based antimalarial compound is a generic DNA binder, a previously unknown property of this antimalarial agent. Similarly to the cocaine-binding aptamer, the ATP-binding aptamer binds two copies of its ligand. But unlike the cocaine-binding aptamer, the ATP aptamer binds its ligand in a cooperative two-site binding manner. In addition, this aptamer must have both sites functional; otherwise, the ligand will bind very weakly. Studies also showed, that if two binding sites are separated, the aptamer becomes more structured and stable, and binding model switches from cooperative to independent for adenosine but not ATP. | |
| dc.identifier.uri | http://hdl.handle.net/10315/37733 | |
| dc.language | en | |
| dc.rights | Author owns copyright, except where explicitly noted. Please contact the author directly with licensing requests. | |
| dc.subject | Biochemistry | |
| dc.subject.keywords | Aptamers | |
| dc.subject.keywords | Isothermal titration calorimetry | |
| dc.subject.keywords | Cocaine-binding aptamer | |
| dc.subject.keywords | ATP-binding aptamer | |
| dc.subject.keywords | Quinine analogues | |
| dc.subject.keywords | Antimalarial compounds | |
| dc.subject.keywords | Independent and cooperative binding models | |
| dc.subject.keywords | Dangling nucleotide | |
| dc.title | Thermodynamic Analysis of Aptamer-Ligand Binding by Isothermal Titration Calorimetry | |
| dc.type | Electronic Thesis or Dissertation |
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