Defining a Stem Length-Dependent Binding Mechanism for the Cocaine-Binding Aptamer. A Combined NMR and Calorimetry Study

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• 作者：Miguel A. D. Neves ; Oren Reinstein ; Philip E. Johnson
• 刊名：Biochemistry
• 出版年：2010
• 出版时间：October 5, 2010
• 年：2010
• 卷：49
• 期：39
• 页码：8478-8487
• 全文大小：1070K
• 年卷期：v.49,no.39(October 5, 2010)
• ISSN：1520-4995

文摘

We have used a combined approach of NMR spectroscopy and isothermal titration calorimetry (ITC) to determine the ligand-binding mechanism employed by a cocaine-binding aptamer. We found that the length of the stem containing the 3′ and 5′ termini determines the nature of the binding mechanism. When this stem is six base pairs long, the secondary structure of the aptamer is fully folded in the free form and only putative tertiary interactions form with ligand binding. If this stem is shortened by three base pairs, the free form of the aptamer contains little secondary structure, and ligand binding triggers secondary structure formation and folding. This binding mechanism is supported by both NMR spectral changes and the ITC measured heat capacity of binding (ΔC_p°). For the aptamer with the long stem the ΔC_p° value is −557 ± 29 cal mol⁻¹ K⁻¹ and for the aptamer with the short stem the ΔC_p° value is −922 ± 51 cal mol⁻¹ K⁻¹. Chemical shift perturbation data and the observation of intermolecular NOEs indicate that the three-way junction is the site of ligand binding.