Biophysics Institute, Johannes Kepler University Linz, Linz, Austria.
Biophys J. 2011 Oct 5;101(7):1781-7. doi: 10.1016/j.bpj.2011.07.054.
Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k(off), compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.
当用于原子力显微镜传感应用时，凝血酶适体的结合强度和稳定性取决于立体参数。为了实现高亲和力结合，我们对连接化学物质进行了立体改进。为此，我们使用两种增强型生物素化凝血酶适体（BFF 和 BFA）通过链霉亲和素固定在原子力显微镜尖端上，应用单分子力谱进行了研究。BFF 由两个通过靠近生物素化末端的互补序列连接在一起的单链适体（aptabody）组成的二聚体。相比之下，BFA 由一条单链 DNA 和支持生物素化部分的互补链组成。通过在力-距离循环中改变拉伸速度，在不同的力加载下使形成的凝血酶-适体复合物断裂，从而可以确定能量景观。结果表明，与 BFF 相比，BFA 适体在研究的加载速率下表现出更高的结合力和明显更低的解离速率常数 k（off）。此外，还可以清楚地证明 aptabody BFF 形成二价复合物的潜力。
