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Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry
Published on: August 23, 2022
Conformational dynamics of the tetracycline-binding aptamer
Ute Förster1, Julia E Weigand, Peter Trojanowski
1Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt, Max-von-Laue-Straße 7, Germany.
Nucleic Acids Research
|November 5, 2011
Summary
Ligand binding to tetracycline aptamers follows a fast two-step mechanism. Mutations altering key bases, like A9, significantly slow binding by disrupting aptamer structure and pre-organization.
Area of Science:
- Molecular Biology
- Biophysics
- Biochemistry
Background:
- Tetracycline-binding aptamers are crucial for regulating gene expression.
- Understanding aptamer conformational dynamics during ligand binding is key to their function.
Purpose of the Study:
- To investigate the conformational dynamics of tetracycline-binding aptamers upon ligand interaction.
- To elucidate the mechanism of ligand binding and the role of specific bases in aptamer function and regulation.
Main Methods:
- Stopped-flow fluorescence spectroscopy to monitor rapid binding events.
- Time-correlated single photon counting experiments to analyze fluorescence changes.
- Analysis of wild-type and mutant (A9G, A13U, A50U) aptamers.
Main Results:
- A fast, two-step binding mechanism was identified: initial binding followed by aptamer reorganization.
- Direct ligand contact points (A13, A50) are involved in the first binding step.
- Mutation A9G significantly decelerated binding, primarily by increasing the reverse reaction rate, indicating loss of tertiary structure and pre-organization.
Conclusions:
- A9 is critical for maintaining aptamer tertiary structure and facilitating efficient ligand binding.
- Aptamer pre-organization is essential for the overall binding process and function.
- Mutations in key regulatory bases can profoundly impact aptamer dynamics and binding kinetics.
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