Improving Thermodynamic Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Incorporation of 8‑trifluoromethyl-2′-deoxyguanosine

In this study, we incorporated 8-trifluoromethyl-2′-deoxyguanosine (FG) into a thrombin binding aptamer (TBA). Circular dichroism, nuclear magnetic resonance (NMR), electrophoresis, and prothrombin time (PT) assay were performed to investigate the structure, thermodynamic stability, biological stability, and anticoagulant activity of the FG-modified TBA sequences. We found that the replacement of FG into TBA sequences led to a remarkable improvement in the melting temperature up to 30 °C compared with the native sequence. The trifluoromethyl group allowed us to investigate the TBA G-quadruplex structure by 19F NMR spectroscopy. Furthermore, PT assays showed that the modified sequences can significantly improve the anticoagulant activity in comparison with the native TBA. Finally, we demonstrated that the trifluoromethyl-modified TBA sequence could function as an anticoagulant reagent in live rats. Our results strongly suggested that FG is a powerful nucleoside derivative to increase the thermodynamic stability and anticoagulant activity of TBA.本研究将8-三氟甲基-2′-脱氧鸟苷（8-trifluoromethyl-2′-deoxyguanosine，FG）整合至凝血酶结合适配体（thrombin binding aptamer，TBA）中。随后采用圆二色谱、核磁共振（nuclear magnetic resonance，NMR）、电泳及凝血酶原时间（prothrombin time，PT）测定等实验手段，对FG修饰TBA序列的结构、热力学稳定性、生物稳定性与抗凝活性进行了系统表征与分析。研究发现，将FG引入TBA序列后，相较未修饰的天然序列，其解链温度显著提升，最高增幅可达30℃。三氟甲基基团可借助19F核磁共振波谱法对TBA的G-四链体结构开展研究。此外，PT测定结果表明，经修饰的序列相较野生型TBA，其抗凝活性显著增强。最后，本研究证实三氟甲基修饰的TBA序列可在活体大鼠体内作为抗凝试剂发挥作用。本研究结果充分表明，FG是一种可有效提升TBA热力学稳定性与抗凝活性的强效核苷衍生物。