title: Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein.
abstract: 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.
authors.affiliation: Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology , 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Amano
authors.affiliation: Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology , 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Takada
authors.affiliation: Facility for RI Research and Education, Instrumental Analysis Center, Yokohama National University , 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan.
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: 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.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology , 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
authors.forename: Gota
authors.initials: G
authors.lastname: Kawai
authors.affiliation: Research Institute for Clinical Oncology , Saitama Cancer Center, Ina, Saitama 362-0806, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kozu
authors.affiliation: Department of Life and Environmental Sciences, Faculty of Engineering, Chiba Institute of Technology , 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
authors.forename: Taiichi
authors.initials: T
authors.lastname: Sakamoto
mesh_terms: D000595:Amino Acid Sequence; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002151:Calorimetry / Q000379:methods; D050676:Core Binding Factor Alpha 2 Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D000072417:Protein Domains; D055672:Static Electricity; D020349:Surface Plasmon Resonance; D013816:Thermodynamics
chemical_list: D052157:Aptamers, Nucleotide; D050676:Core Binding Factor Alpha 2 Subunit; D008024:Ligands; C493728:RUNX1 protein, human