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Characterization of enhanced monovalent and bivalent thrombin DNA aptamer binding using single molecule force
Isabel Neundlinger1, Alexandra Poturnayova, Ivana Karpisova
1Biophysics Institute, Johannes Kepler University Linz, Linz, Austria.
Biophysical Journal
|October 4, 2011
Summary
The study investigated thrombin aptamers for atomic force microscopy, finding that the BFA aptamer exhibits stronger binding and greater stability than the BFF aptamer. These findings advance aptamer-based sensing technologies.
Area of Science:
- Biochemistry
- Biophysics
- Materials Science
Background:
- Thrombin aptamers are crucial for atomic force microscopy (AFM) sensing applications.
- Optimizing aptamer sterical parameters enhances binding affinity and stability.
- Linker chemistry modifications are key to high-affinity aptamer-based binding.
Purpose of the Study:
- To investigate the impact of sterical parameters on thrombin aptamer binding strength and stability for AFM sensing.
- To compare the performance of two distinct thrombin aptamer structures, BFF and BFA, in high-affinity binding applications.
- To characterize the energy landscape of thrombin-aptamer interactions using single molecule force spectroscopy.
Main Methods:
- Single molecule force spectroscopy (SMFS) was employed to analyze thrombin-aptamer interactions.
- Two enhanced biotinylated thrombin aptamers, BFF (dimer) and BFA (single strand with complementary part), were immobilized on AFM tips via streptavidin.
- Force-distance cycles with varying pulling velocities were used to rupture thrombin-aptamer complexes and determine binding parameters.
Main Results:
- The BFA aptamer demonstrated a higher binding force compared to the BFF aptamer across investigated loading rates.
- The dissociation rate constant (k(off)) for the BFA aptamer was significantly lower than that of the BFF aptamer, indicating greater stability.
- The study confirmed the potential of the aptabody BFF to form bivalent complexes, although BFA showed superior binding performance.
Conclusions:
- Sterical improvements in linker chemistry significantly enhance thrombin aptamer binding affinity for AFM applications.
- The BFA aptamer structure offers superior binding force and stability compared to the BFF aptamer for thrombin detection.
- This research provides valuable insights into aptamer design for optimizing molecular recognition and force spectroscopy measurements.
