Ames Laboratory, US DOE, Ames, IA, USA; Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Ames, IA, USA.
Methods. 2013 Sep 15;63(2):178-87. doi: 10.1016/j.ymeth.2013.03.016. Epub 2013 Mar 25.
Aptamers are short, single-stranded nucleic acids with structures that frequently change upon ligand binding and are sensitive to the ionic environment. To achieve facile application of aptamers in controlling cellular activities, a better understanding is needed of aptamer ligand binding parameters, structures, intramolecular mobilities and how these structures adapt to different ionic environments with consequent effects on their ligand binding characteristics. Here we discuss the integration of biochemical analysis with NMR spectroscopy and computational modeling to explore the relation between ligand binding and structural malleability of some well-studied aptamers. Several methods for determining aptamer binding affinity and specificity are discussed, including isothermal titration calorimetry, steady state fluorescence of 2-aminopurine substituted aptamers, and dye displacement assays. Also considered are aspects of molecular dynamics simulations specific to aptamers including adding ions and simulating aptamer structure in the absence of ligand when NMR spectroscopy or X-ray crystallography structures of the unoccupied aptamer are not available. We focus specifically on RNA aptamers that bind small molecule ligands as would be applied in sensors or integrated into riboswitches such as to measure the products of metabolic activity.
适体是短的、单链的核酸，其结构在配体结合时经常发生变化，并且对离子环境敏感。为了使适体在控制细胞活动方面能够方便地应用，需要更好地了解适体配体结合参数、结构、分子内迁移率以及这些结构如何适应不同的离子环境，从而对其配体结合特性产生影响。在这里，我们讨论了将生化分析与 NMR 光谱和计算建模相结合，以探索一些研究充分的适体的配体结合与结构柔韧性之间的关系。讨论了几种确定适体结合亲和力和特异性的方法，包括等温滴定量热法、2-氨基嘌呤取代适体的稳态荧光法和染料置换测定法。还考虑了适体分子动力学模拟的特定方面，包括添加离子和在没有配体的情况下模拟适体结构，当 NMR 光谱或 X 射线晶体学结构不可用时。我们特别关注与小分子配体结合的 RNA 适体，这些适体可用于传感器或整合到核糖开关中，以测量代谢活性的产物。
