Characterizing the Interactions of Aptamers with their Ligands using Isothermal Titration Calorimetry and Fluorescence Spectrometry

Aptamers are short single-stranded DNA or RNA molecules that are selected to bind a target often with high affinity and specificity. Some of these targets include small molecules, proteins, nucleic acids, cells and tissues. Isothermal Titration Calorimetry (ITC) and Fluorescence Spectrometry were employed to characterize the binding interactions of aptamers with their ligands. This dissertation comprises two separate research projects; in the first project, ITC was used to explore how the binding affinity of structure-switching aptamers toward their ligand varies when altering the NaCl concentration. It was shown that the binding affinity of MN19, a variant of the cocaine-binding aptamer, decreases when the NaCl concentration is increased from 140 mM to 1000 mM. The affinity increases again when the NaCl concentration is increased to 2000 mM.

The next project showcases the use of fluorescence spectrometry to characterize the interactions of methylene blue with different aptamers. Methylene blue is a redox-reporter which is used in Electrochemical aptamer-based biosensing platforms. It was shown that methylene blue binds to the MN19 aptamer, and therefore, it was of interest to see if methylene blue binds to other aptamers and to explore any similarities in their aptamer structures. Fluorescence spectrometry was used to see if methylene blue binds to other aptamers by measuring the change in fluorescence intensity of methylene blue and if binding occurred, the binding affinity was quantified. It was determined that methylene blue may be interacting and binding tightly to DNA aptamer structures which includes bulges and stem-loops but may be binding very weakly to duplex DNA such as the Dickerson Drew Dodecamer and a Hexamer.