Single-molecule measurements of the binding between small molecules and DNA aptamers.
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 (ΔG(unfold)) between these two aptamers yields a ΔG 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 μM, a value consistent with the K(d) obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 μM) and close to that determined by affinity chromatography in the literature (6 ± 3 μM). 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.
Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44242, USA.
Philip M
PM
Yangyuoru
Soma
S
Dhakal
Zhongbo
Z
Yu
Deepak
D
Koirala
Simon M
SM
Mwongela
Hanbin
H
Mao
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019075:Electrophoresis, Capillary; D008024:Ligands; D055595:Mechanical Phenomena; D052898:Optical Tweezers; D013816:Thermodynamics
D052157:Aptamers, Nucleotide; D008024:Ligands; D000255:Adenosine Triphosphate