Structural Effects of Modifications on Interactions of Thrombin Binding Aptamer with Thrombin: A Molecular Dynamic Study

Thrombin is a protease that promotes the formation of blood clots by converting fibrinogen to fibrin. Understanding biophysical properties such as structure and interactions with other molecules is important for designing new thrombin inhibitors used to treat blood clotting disorders requiring anticoagulation therapy. Thrombin inhibitors include TBA and HD22 aptamer which are oligonucleotides that bind thrombin and prevent blood coagulation. These aptamers form stable G-quadruplexes that bind thrombin in its exosites. TBA was a promising target for anticoagulation therapy but it requires high dosage values to be clinically effective. Several approaches have been used to improve its efficacy as an anticoagulant. In this study we are investigating the effects of modifying the aptamer on its interactions with thrombin. Specifically, we hypothesized that dendrimer modifications to the 5’ end of the TBA, and NHS (N-hydroxysuccinimide)-carboxy in the thymines in the loop region of the quadruplex, could improve its binding affinity by providing additional interactions with Thrombin. Circular Dichroism (CD) spectra and surface plasmon resonance (SPR) show that while the modifications are well tolerated in the aptamer, they do not result in an increase in thrombin binding affinity. In this study we employ all-atom molecular dynamic (MD) simulations to understand the structural effects of the modifications and propose sites for modifications that can boost the interactions of TBA with thrombin.