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Biochemistry. 2016 Nov 15;55(45):6221-6229. Epub 2016 Nov 1.

Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein.

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Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology , 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
Facility for RI Research and Education, Instrumental Analysis Center, Yokohama National University , 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo , Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Ribomic Inc. , 3-16-13 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan.
Research Institute for Clinical Oncology , Saitama Cancer Center, Ina, Saitama 362-0806, Japan.


AML1 (RUNX1) protein is an essential transcription factor involved in the development of hematopoietic cells. Several genetic aberrations that disrupt the function of AML1 have been frequently observed in human leukemia. AML1 contains a DNA-binding domain known as the Runt domain (RD), which recognizes the RD-binding double-stranded DNA element of target genes. In this study, we identified high-affinity RNA aptamers that bind to RD by systematic evolution of ligands by exponential enrichment. The binding assay using surface plasmon resonance indicated that a shortened aptamer retained the ability to bind to RD when 1 M potassium acetate was used. A thermodynamic study using isothermal titration calorimetry (ITC) showed that the aptamer-RD interaction is driven by a large enthalpy change, and its unfavorable entropy change is compensated by a favorable enthalpy change. Furthermore, the binding heat capacity change was identified from the ITC data at various temperatures. The aptamer binding showed a large negative heat capacity change, which suggests that a large apolar surface is buried upon such binding. Thus, we proposed that the aptamer binds to RD with long-range electrostatic force in the early stage of the association and then changes its conformation and recognizes a large surface area of RD. These findings about the biophysics of aptamer binding should be useful for understanding the mechanism of RNA-protein interaction and optimizing and modifying RNA aptamers.

[Indexed for MEDLINE]

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