Studying small molecule-aptamer interactions using MicroScale Thermophoresis (MST)
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Aptamers are potent and versatile binding molecules recognizing various classes of target molecules. Even challenging targets such as small molecules can be identified and bound by aptamers. Studying the interaction between aptamers and drugs, antibiotics or metabolites in detail is however difficult due to the lack of sophisticated analysis methods. Basic binding parameters of these small molecule-aptamer interactions such as binding affinity, stoichiometry and thermodynamics are elaborately to access using the state of the art technologies. The innovative MicroScale Thermophoresis (MST) is a novel, rapid and precise method to characterize these small molecule-aptamer interactions in solution at microliter scale. The technology is based on the movement of molecules through temperature gradients, a physical effect referred to as thermophoresis. The thermophoretic movement of a molecule depends – besides on its size – on charge and hydration shell. Upon the interaction of a small molecule and an aptamer, at least one of these parameters is altered, leading to a change in the movement behavior, which can be used to quantify molecular interactions independent of the size of the target molecule. The MST offers free choice of buffers, even measurements in complex bioliquids are possible. The dynamic affinity range covers the pM to mM range and is therefore perfectly suited to analyze small molecule-aptamer interactions. This section describes a protocol how quantitative binding parameters for aptamer-small molecule interactions can be obtained by MST. This is demonstrated by mapping down the binding site of the well-known ATP aptamer DH25.42 to a specific region at the adenine of the ATP molecule.
Copyright © 2015 Elsevier Inc. All rights reserved.
Binding parameters; MicroScale Thermophoresis (MST); Small molecule–aptamer interactions
- PMID: 26334574; DOI:10.1016/j.ymeth.2015.08.023
Aptamers are short single stranded nucleic acids, possessing characteristics of antibodies. They bind to ligands with high affinity and specificity. Aptamers usually recognize the 3D structure of the target molecule they are selected for. Systematic evolution of ligands by exponential enrichment (SELEX) is an in vitro evolution process, in which the aptamers are raised (1-2). In the beginning of this process, a large oligonucleotide library is incubated with the target of interest, which can be a small molecule or even a cell. By exclusion methods, such as affinity chromatography, unbound nucleic acids are removed. Bound sequences are eluted and then amplified by PCR for subsequent selection rounds. Known 5´and 3´ sequences attached to the oligonucleotide library serve as PCR primers. By increasing the stringency and inclusion of counter-SELEX rounds, an aptamer can be taught to bind a specific target and to avoid binding of another unwanted target. After a suitable number of selection rounds, bound oligonucleotides are sequenced and relevant binders are identified bioinformaticaly.
Upon successful selection, aptamer candidates are characterized in terms of essential binding parameters, such as binding affinity. However, an analytical bottleneck slows down the development of aptamers (7-25 kDa) towards small molecules (0.1-1 kDa), as many state-of-the-art technologies lack the necessary sensitivity to elucidate binding parameters of these critical interactions. Main issue is the small size of both binding partners accompanied with only marginal changes in size and structure upon binding of the partners. Furthermore, buffer restrictions, large sample consumption, surface immobilization and mass transport limitations have to be considered.
MicroScale Thermophoresis (MST) may fill the gap in analytical methods, as it is working independently of size of interaction partners. The technology monitors movement of molecules in temperature gradients (thermophoresis), which relies on size, charge and hydration-shell. Formation of a complex upon binding, results in different thermophoresis and can be used to determine basic binding parameter of the molecular interaction. Interactions between smallest interaction partners can be characterized without restrictions in data quality. In addition, MST works in solution at low sample consumption and freedom of buffer usage.
To demonstrate the capabilities of this technology in aptamer characterization, the current study describes the mapping of the binding site of the well-studied aptamer D25.42 on its binding partner ATP (3). Analyzing the binding behavior of different ATP derivatives by MST, the aptamer-interaction site on ATP was determined being the adenine group.
Importance of this study: The study underlines the great analytical potential of the MicroScale Thermophoresis. The technology allows to study the interaction of even smallest interaction partners (aptamer D25.42-Cy5; 7.8 kDa and Adenine; 0.13 kDa) without restrictions in data quality. MicroScale Thermophoresis may serve the analytical need in the characterization process of aptamers selected to small molecules.
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- David E. Huizenga, Jack W. Szostak (1995) A DNA Aptamer That Binds Adenosine and ATP Biochemistry, 1995, 34 (2), pp 656–665
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