Thermodynamic and Kinetic Analysis of Aptamer-Ligand Interactions Using Isothermal Titration Calorimetry

Aptamers are short single-stranded DNA or RNA oligonucleotide sequences capable of binding to a broad range of target molecules with high affinity and specificity. They can interact with a large variety of targets such as small molecules, ions, enzymes, and proteins employing all kinds of non-covalent interactions. Isothermal Titration Calorimetry was employed to explore the binding behaviour of aptamer-ligand interactions using the cocaine-binding aptamer as a model system. This dissertation is an assembly of two distinct research projects; In the first part, a bifunctional cocaine and deoxycholic acid-binding aptamer was constructed from individual cocaine-binding aptamer variants and the binding affinity and thermodynamics were measured using isothermal titration calorimetry. We show that the bifunctional aptamer binds its ligands with positive cooperativity, having a Hill coefficient of 1.2 -1.5, whether the ligands are added individually or as an equimolar mixture. A mechanism where dynamics at one ligand-binding site is affected by the presence of the ligand at the other is proposed to account for the cooperative binding.

The next chapter highlights the use of kinITC to extract binding rates from ITC experiments. Determining the binding thermodynamics and kinetics between aptamers and ligands is important for understanding their recognition mechanisms as well as providing a framework for developing biosensors. One aptamer that has been particularly well studied is the cocaine-binding aptamer. There has been a lot of research conducted on the thermodynamics for the cocaine-binding aptamer however, one feature not reported yet is the binding kinetics. Here, we measure the kinetics of quinine binding to two sequence variants of the cocaine-binding aptamer, one with a short stem-one (MN19) and the other with a long stem-one (MN4). When stem-one of the aptamer is six base pairs long, the aptamer retains its secondary structure in its free and bound forms. When the length is shortened to three base pairs, such as for MN19, the aptamer is loosely folded in its unbound state and undergoes structural changes upon binding quinine. Both binding kinetics and thermodynamic data were acquired as a function of different temperatures. A 1:1 binding model was applied to extract binding rates for both variants of the cocaine-binding aptamer. Arrhenius plots were derived to compare the activation energies for MN4 to that of MN19 since MN19 exhibits ligand-dependent conformational changes. The values obtained for the transition state enthalpy barrier were averaged to be 5.1 kcal mol^(-1) for MN4 and -0.8 kcal mol^(-1) for MN19. These results suggest that MN19 has an energy barrier that is almost negligible under standard conditions.

Aptamers are short single-stranded DNA or RNA oligonucleotide sequences capable of binding to a broad range of target molecules with high affinity and specificity. They can interact with a large variety of targets such as small molecules, ions, enzymes, and proteins employing all kinds of non-covalent interactions. Isothermal Titration Calorimetry was employed to explore the binding behaviour of aptamer-ligand interactions using the cocaine-binding aptamer as a model system. This dissertation is an assembly of two distinct research projects; In the first part, a bifunctional cocaine and deoxycholic acid-binding aptamer was constructed from individual cocaine-binding aptamer variants and the binding affinity and thermodynamics were measured using isothermal titration calorimetry. We show that the bifunctional aptamer binds its ligands with positive cooperativity, having a Hill coefficient of 1.2 -1.5, whether the ligands are added individually or as an equimolar mixture. A mechanism where dynamics at one ligand-binding site is affected by the presence of the ligand at the other is proposed to account for the cooperative binding.

The next chapter highlights the use of kinITC to extract binding rates from ITC experiments. Determining the binding thermodynamics and kinetics between aptamers and ligands is important for understanding their recognition mechanisms as well as providing a framework for developing biosensors. One aptamer that has been particularly well studied is the cocaine-binding aptamer. There has been a lot of research conducted on the thermodynamics for the cocaine-binding aptamer however, one feature not reported yet is the binding kinetics. Here, we measure the kinetics of quinine binding to two sequence variants of the cocaine-binding aptamer, one with a short stem-one (MN19) and the other with a long stem-one (MN4). When stem-one of the aptamer is six base pairs long, the aptamer retains its secondary structure in its free and bound forms. When the length is shortened to three base pairs, such as for MN19, the aptamer is loosely folded in its unbound state and undergoes structural changes upon binding quinine. Both binding kinetics and thermodynamic data were acquired as a function of different temperatures. A 1:1 binding model was applied to extract binding rates for both variants of the cocaine-binding aptamer. Arrhenius plots were derived to compare the activation energies for MN4 to that of MN19 since MN19 exhibits ligand-dependent conformational changes. The values obtained for the transition state enthalpy barrier were averaged to be 5.1 kcal mol^(-1) for MN4 and -0.8 kcal mol^(-1) for MN19. These results suggest that MN19 has an energy barrier that is almost negligible under standard conditions.