title: 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.
authors.affiliation: Biophysics Institute, Johannes Kepler University Linz, Linz, Austria.
authors.forename: Isabel
authors.initials: I
authors.lastname: Neundlinger
authors.forename: Alexandra
authors.initials: A
authors.lastname: Poturnayova
authors.forename: Ivana
authors.initials: I
authors.lastname: Karpisova
authors.forename: Christian
authors.initials: C
authors.lastname: Rankl
authors.forename: Peter
authors.initials: P
authors.lastname: Hinterdorfer
authors.forename: Maja
authors.initials: M
authors.lastname: Snejdarkova
authors.forename: Tibor
authors.initials: T
authors.lastname: Hianik
authors.forename: Andreas
authors.initials: A
authors.lastname: Ebner
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019281:Dimerization; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods*; D011485:Protein Binding; D019809:Streptavidin / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D004800:Enzymes, Immobilized; D019809:Streptavidin; D013917:Thrombin