Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force spectroscopy.

ABSTRACT: 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.

SUBMITTER: Neundlinger I 

PROVIDER: S-EPMC3183820 | biostudies-other | 2011 Oct

REPOSITORIES: biostudies-other