Single-Molecule Measurements of the Binding between Small Molecules and DNA Aptamers

Abstract

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.

Direct Measurement of Through‐Bond Effects in Molecular Multivalent Interactions

“…[34,35] It is noted that Mao et al use optical tweezers to determine the dissociation constant for aptamer-ligandi nteractions through measuring aptamer unfolding force (21~38 pn). [36] However,t heir values were much lower than above unbinding forces. The comparison of these force values indicates that the rupture events in our FIRMS experiments are likely due to the direct unbindingo ft he aptamer-thrombin complex instead of unfolding of the aptamer.…”

“…At hrough-bond effect in multivalent interactions has been proposed to reveal the subtle influence of the protein dynamics and the perturbation exertedb yt he presence of al igand at one exosite. [37][38][39][40][41][42] The case of thrombin particularly represents a typical example of how ligand bindingt om ore than one exosite regulates enzymatic activity.A lthough force spectroscopy has been used to investigate the interactions between thrombin and its aptamers, [34,36,43,44] the through-bond effects were unresolved in force spectra. It remains unknown whether the binding force of the thrombin-aptamer complex will alter with the presence of secondary aptamer, which may be valuable for understanding the affinity and selectivity of high-performance ligands.…”

Mechanochemical Sensing: A Biomimetic Sensing Strategy

“…[38] With this SMMS strategy,t he binding constant (K d )b etween an aptamer and as mall molecule, ATP, was successfully measured in optical tweezers. [38] The K d was estimated from ac hange in the free energy of the binding between the ATPa nd the aptamers (DG binding )b yu sing the relationship DG binding = ÀRTlnK d , where R is the gasc onstant and T is temperature. As the change in free energy is as tate function, aH ess-like cycle was used to retrieve the DG binding value (Figure 5d).…”

RNA and DNA Diagnostics

“…However, this microfluidic mechanochemical strategy can function as a highly sensitive generic biosensor after incorporation of specific recognition element such as an aptamer. Double-headed arrows depict the average time observed before the hopping ceases to the unfolded hairpin state, which indicates the binding of the SNP to the hairpin DNA aptamer can be used as a basic biosensing platform to evaluate the binding constant (K d ) between an aptamer and a small molecule such as ATP using laser tweezers (Yangyuoru et al 2012). This technique offers a significant advantage to determine the dissociation constant (K d ) for aptamer-ligand interactions at the single-molecule level from only one ligand concentration (Nguyen et al 2011).…”