Thermodynamic Analysis of Aptamer-Ligand Binding by Isothermal Titration Calorimetry

dc.contributor.advisorJohnson, Philip E.
dc.contributor.authorSlavkovic, Sladjana
dc.date.accessioned2020-08-11T12:47:28Z
dc.date.available2020-08-11T12:47:28Z
dc.date.copyright2020-04
dc.date.issued2020-08-11
dc.date.updated2020-08-11T12:47:28Z
dc.degree.disciplineChemistry
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractAptamers 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.urihttp://hdl.handle.net/10315/37733
dc.languageen
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectBiochemistry
dc.subject.keywordsAptamers
dc.subject.keywordsIsothermal titration calorimetry
dc.subject.keywordsCocaine-binding aptamer
dc.subject.keywordsATP-binding aptamer
dc.subject.keywordsQuinine analogues
dc.subject.keywordsAntimalarial compounds
dc.subject.keywordsIndependent and cooperative binding models
dc.subject.keywordsDangling nucleotide
dc.titleThermodynamic Analysis of Aptamer-Ligand Binding by Isothermal Titration Calorimetry
dc.typeElectronic Thesis or Dissertation

Files

Original bundle
Now showing 1 - 1 of 1
Loading...
Thumbnail Image
Name:
Slavkovic_Sladjana_2020_PhD.pdf
Size:
7.56 MB
Format:
Adobe Portable Document Format
License bundle
Now showing 1 - 2 of 2
No Thumbnail Available
Name:
license.txt
Size:
1.83 KB
Format:
Plain Text
Description:
No Thumbnail Available
Name:
YorkU_ETDlicense.txt
Size:
3.36 KB
Format:
Plain Text
Description:

Collections