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.

Potential uses of G-quadruplex-forming aptamers

“…In addition, these dimers demonstrated levels of fibrinogen cleavage inhibition in thrombin which were almost twice as high as that of monomolecular aptamers (Ponikova et al 2011). The existence of two binding sites in the dimers was also confirmed by single molecule force spectroscopy (Neundlinger et al 2011), emphasizing that the substantial structural flexibility of aptamers is an additional advantage that makes these molecules excellent candidates for use as recognition elements in biosensors.…”

Single molecular recognition force spectroscopy study of a DNA aptamer with the target epithelial cell adhesion molecule

“…The system has more attempts to make the transition at a very slow force loading rate 25 than at faster force loading rates. This behavior can be described with the so-called Bell-Evans model.…”

“…High expression levels of EpCAM usually is associated with advanced stages of disease and worse overall survival, e.g. in breast cancer and ovarian cancer as well as in 25 pancreatic, urothelial, and gallbladder carcinoma. 5,6 In addition, because of its tumor specific overexpression EpCAM is an attractive target for tumor diagnosis and therapy.…”

“…1. The prepared silicon substrates functionalized with EpCAM were 25 characterized by AFM. Topographical images were taken in PBS at room temperature.…”

Atomic force microscopy fishing and mass spectrometry identification of gp120 on immobilized aptamers

“…AFM is broadly used due to its wide capabilities for determination of the interaction force between the tip-tethered aptamer and a protein immobilized on the substrate surface, [19][20][21] and also for biospecific recognition of protein on the surface. 22,23 The most popular object of aptamer-based applications in molecular research is detection of thrombin. [24][25][26][27] The goal of our research was to highlight the advantages of aptamers as molecular probes for biospecific fishing of gp120 HIV protein from the solution on the AFM chip surface.…”