Studies of the Binding Mechanism between Aptamers and Thrombin by Surface Plasmon Resonance and Circular Dichroism

• Main Authors: Ren-hao chen, 陳人豪
• Other Authors: Wen-yih chen
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/09067407226002566458

碩士 === 國立中央大學 === 化學工程與材料工程研究所 === 98 === This study characterizes the interaction mechanism between aptamers and human thrombin by surface plasmon resonance (SPR) and ciucular dichroism (CD). Thrombin, a multifunctional serine protease, has both procoagulant and anticoagulant functions in human blood. Thrombin involves two electropositive exosites；one is fibrinogen-binding site and the other is heparin-binding site. Two thrombin-binding aptamers have been selected by SELEX technique over the past decade, respectively. One is 15-mer aptamer binds at fibrinogen-binding site of thrombin, while 29-mer aptamer binds at the heparin binding site of thrombin. In the past years many papers have reported the interaction between 15-mer aptamer and thrombin, however the difference of the two aptamers bind to thrombin is still lacking and worth of investigation. In this study, we combined kinetics and conformational information to compare the binding mechanism between these two aptamers with thrombin. Two experiments were mainly performed in this investigation. CD assay demonstrated the comformational feature of different aptamers binding to thrombin, while SPR provided kinetic constant (Ka) in different binding parameters of aqueous solution (salt concentration and pH). From the results, we found that 20-mer aptamer binding to thrombin by G-guadruplex structure and dominated by electrostatic interactions. The 29-mer aptamer binds to thrombin by hairpin structure and is driven by hydrophobic effects. Furthermore, we comfirmed this argument by Isothermal Titration Calometry measurements . By experimental results, we suggested that the structure of these two aptamers is an important factor to cause the different binding mechanism between these two aptamers with thrombin.