Entzian Clemens, Schubert Thomas
2bind GmbH, Josef Engertstraße 13, 93053 Regensburg, Germany.
2bind GmbH, Josef Engertstraße 13, 93053 Regensburg, Germany.
Methods. 2016 Mar 15;97:27-34. doi: 10.1016/j.ymeth.2015.08.023. Epub 2015 Aug 31.
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.
适体是能够识别各类靶分子的强效且多功能的结合分子。即使是小分子这类具有挑战性的靶标，也能被适体识别并结合。然而，由于缺乏先进的分析方法，详细研究适体与药物、抗生素或代谢物之间的相互作用颇具难度。利用现有技术精确获取这些小分子 - 适体相互作用的基本结合参数，如结合亲和力、化学计量学和热力学参数等并非易事。创新的微量热泳技术（MST）是一种新颖、快速且精确的方法，可在微升规模下表征溶液中这些小分子 - 适体的相互作用。该技术基于分子在温度梯度中的移动，这一物理效应被称为热泳。分子的热泳移动除了取决于其大小外，还取决于电荷和水化层。当小分子与适体相互作用时，这些参数中至少有一个会发生改变，从而导致移动行为发生变化，这可用于独立于靶分子大小来量化分子间相互作用。MST可自由选择缓冲液，甚至在复杂生物液体中进行测量也可行。其动态亲和力范围涵盖了从皮摩尔到毫摩尔的范围，因此非常适合分析小分子 - 适体相互作用。本节描述了一种通过MST获取适体 - 小分子相互作用定量结合参数的方案。通过将著名的ATP适体DH25.42的结合位点定位到ATP分子腺嘌呤的特定区域来进行说明。
