Single-Molecule Measurements of the Binding between Small Molecules and DNA Aptamers Philip M. Yangyuoru, ‡ Soma Dhakal, ‡ Zhongbo Yu, ‡ Deepak Koirala, ‡ Simon M. Mwongela, † and Hanbin Mao* ,‡ † School of Science & Technology, Georgia Gwinnett College, Lawrenceville, Georgia 30043, United States ‡ Department of Chemistry and Biochemistry, Kent State University, Kent, Ohio 44242, United States * S Supporting Information 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. T he interaction between nucleic acids and small molecules is continuously generating significant interest due to their widespread use in analytical and bioanalytical applications. Specific nucleic acids such as aptamers have been shown to bind not only to nucleic acids but also to non nucleic acid targets such as small molecules, 1,2 proteins, 3 and cells. 4 Aptamers are single-stranded ligand-binding nucleic acids with affinity and selectivity comparable to those of antibod- ies. 5,6 Compared to antibodies, DNA aptamers are easier to obtain, more stable, and more resistant to biodegradation. As such, they are suitable for demanding conditions such as extreme pH and temperatures. These properties render them ideal candidates for sensors used in medical diagnostics and environmental monitoring. 7 The binding affinity between an aptamer and a ligand is a critical factor to reflect the sensitivity of aptamer-based biosensors. 8 Conventionally, the binding is measured by techniques such as electrochemical detection, 9,10 fluores- cence, 11 colorimetric 12 or surface plasma resonance (SPR) based detection, 13 and capillary electrophoresis (CE) separa- tion, 14-16 among others. 17 At the single-molecular level, atomic force microscopy (AFM) has been recently used to determine the adenosine 5′-triphosphate disodium salt (ATP) binding to an aptamer made of two independently split DNA strands. 18 Single-molecule fluorescence resonance energy transfer (FRET) has also been explored to evaluate the binding of a DNA aptamer with its binding target, vascular endothelial growth factor (VEGF). 19 However, a majority of these methods require a series of experiments with varying ligand or aptamer concentrations to determine the dissociation constant. Apart from being tedious and time-consuming, such experiments are prone to a decreased signal-to-noise ratio due to run-to-run variations. Another disadvantage is that they require a substantial amount of materials. This becomes problematic especially in drug screening processes where the amount of a sample is often limited. As an alternative, here, we employ a laser tweezers instrument to determine the dissociation constant between ATP and a DNA aptamer by using only one concentration of ATP. Developed in the 1980s, 20 laser tweezers have been used extensively to probe the mechanical stabilities not only for DNA 21,22 and RNA 23-26 but also for proteins 27 at the single- molecule level. Recently, we have extended this method to investigate the interaction between a DNA G-quadruplex and its ligand, 28 as well as to serve as a force-based biosensing platform. 29 Here, we use this technique to scrutinize the interaction between aptamers and ligands. The binding of ATP Received: February 13, 2012 Accepted: May 22, 2012 Published: May 23, 2012 Article pubs.acs.org/ac © 2012 American Chemical Society 5298 dx.doi.org/10.1021/ac300427d | Anal. Chem. 2012, 84, 5298-5303