Development of a thermal-stable structure-switching cocaine-binding aptamer

Garaev

Hunter

et al. 2020

Biophysical Journal

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We have used magnetization transfer NMR experiments to measure the exchange rate constant (k ex ) of the imino protons in the unbound, cocaine-bound, and quinine-bound forms of the cocaine-binding DNA aptamer. Both long-stem 1 (MN4) and short-stem 1 (MN19) variants were analyzed, corresponding to structures with a prefolded secondary structure and ligandinduced-folding versions of this aptamer, respectively. The k ex values were measured as a function of temperature from 5 to 45 C to determine the thermodynamics of the base pair opening for MN4. We find that the base pairs close to the ligand-binding site become stronger upon ligand binding, whereas those located away from the binding site do not strengthen. With the buffer conditions used in this study, we observe imino 1 H signals in MN19 not previously seen, which leads us to conclude that in the free form, both stem 2 and parts of stem 3 are formed and that the base pairs in stem 1 become structured or more rigid upon binding. This is consistent with the k ex values for MN19 decreasing in both stem 1 and at the ligand-binding site. Based on the temperature dependence of the k ex values, we find that MN19 is more dynamic than MN4 in the free and both ligand-bound forms. For MN4, ligand-binding results in the reduction of dynamics that are localized to the binding site. These results demonstrate that an aptamer in which the base pairs are preformed also experiences a reduction in dynamics with ligand binding.

Section: Discussionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 93%

Reduction in Dynamics of Base pair Opening upon Ligand Binding by the Cocaine-Binding Aptamer

Garaev

Hunter

et al. 2020

Biophysical Journal

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“…5 ). The DNA UV absorbance at 260 nm of unbound MN19 aptamer does not show a shift observed previously 31 , and as expected for a loosely folded or unfolded nucleic acid molecule. For free MN19, the UV absorption increases linearly with temperature (Fig.…”

Section: Resultssupporting

confidence: 86%

DNA binding by the antimalarial compound artemisinin

Slavkovic

Shoara

et al. 2022

Sci Rep

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Artemisinin (ART) is a vital medicinal compound that is used alone or as part of a combination therapy against malaria. ART is thought to function by attaching to heme covalently and alkylating a range of proteins. Using a combination of biophysical methods, we demonstrate that ART is bound by three-way junction and duplex containing DNA molecules. Binding of ART by DNA is first shown for the cocaine-binding DNA aptamer and extensively studied using this DNA molecule. Isothermal titration calorimetry methods show that the binding of ART is both entropically and enthalpically driven at physiological NaCl concentration. Native mass spectrometry methods confirm DNA binding and show that a non-covalent complex is formed. Nuclear magnetic resonance spectroscopy shows that ART binds at the three-way junction of the cocaine-binding aptamer, and that binding results in the folding of the structure-switching variant of this aptamer. This structure-switching ability was exploited using the photochrome aptamer switch assay to demonstrate that ART can be detected using this biosensing assay. This study is the first to demonstrate the DNA binding ability of ART and should lay the foundation for further work to study implications of DNA binding for the antimalarial activity of ART.

“…( 1 ): where [L] is the ligand concentration, X is the fraction bound, n equals 2 binding events and K d is the dissociation constant at 20 °C as determined in this study. To quantify the thermal shift, the first derivative of each thermal curve was plotted as a function of temperature using OriginPro 2016 software (OriginLab Corporation, Northhampton, MA, USA), as described previously 34 .…”

Section: Methodsmentioning

confidence: 99%

Thermodynamic analysis of cooperative ligand binding by the ATP-binding DNA aptamer indicates a population-shift binding mechanism

Slavkovic

Zhu

et al. 2020

Sci Rep

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The ATP-binding DNA aptamer is often used as a model system for developing new aptamer-based biosensor methods. This aptamer follows a structure-switching binding mechanism and is unusual in that it binds two copies of its ligand. We have used isothermal titration calorimetry methods to study the binding of ATP, ADP, AMP and adenosine to the ATP-binding aptamer. Using both individual and global fitting methods, we show that this aptamer follows a positive cooperative binding mechanism. We have determined the binding affinity and thermodynamics for both ligand-binding sites. By separating the ligand-binding sites by an additional four base pairs, we engineered a variant of this aptamer that binds two adenosine ligands in an independent manner. Together with NMR and thermal stability experiments, these data indicate that the ATP-binding DNA aptamer follows a population-shift binding mechanism that is the source of the positive binding cooperativity by the aptamer.