Journal
ANALYTICAL CHEMISTRY
Volume 84, Issue 12, Pages 5298-5303Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac300427d
Keywords
-
Categories
Funding
- National Science Foundation [CHE-1026532]
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [1026532] Funding Source: National Science Foundation
Ask authors/readers for more resources
Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5'-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (Delta G(unfold)) between these two aptamers yields a Delta G of 33 +/- 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (K-d) of 2.0 +/- 0.2 mu M, a value consistent with the K-d obtained from our equilibrated capillary electrophoresis (CE) (2.4 +/- 0.4 mu M) and close to that determined by affinity chromatography in the literature (6 +/- 3 mu M). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available