A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis
A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis
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DOI：
10.1039/c0an00842g
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发表时间：
2011-01-01
期刊：
影响因子：
4.2
通讯作者：
Easley, Christopher J.
中科院分区：
文献类型：
--
作者：
Hu, Jiaming;Easley, Christopher J.
自动微芯片电泳是一种简单快速的方法，可以测量适配体对大分子和小分子靶标的有效解离常数(K（d),(eff)）。选择人凝血酶、免疫球蛋白E （IgE）和三磷酸腺苷（ATP）作为模型分析物来验证该方法，使用四种配体，包括两个凝血酶DNA适体（两个不同的表位）、一个IgE适体和一个ATP适体。该方法是基于微芯片版本的DNA迁移转移测定。当以大分子为靶标时，非变性微芯片凝胶电泳分离的DNA可以从目标结合的适体中分离和定量。为了将该技术扩展到小分子靶标，如ATP，使用了适体/竞争体策略，其中与适体互补的DNA竞争体可以被ATP取代并电泳分解。使用自动微芯片电泳平台，11个滴定样品的平行分离在大约0.5小时内完成。分析性能比较表明，与之前报道的适体K(d)测量方法相比，我们的方法在最小化试剂消耗（通常为数十pmol的适体和目标），减少分析时间和最小化用户交互方面具有显著优势。此外，适配体针对大小目标的K（d,eff）测量的灵活性和便利性使其成为一种独特而有价值的方法，应该得到广泛使用。最后，在适体选择过程（例如SELEX）中使用该方法的可行性通过对已知凝血酶适体（THRaptA）进行精确的体积K（d,eff）测量，以低至总库的5.0%（摩尔%）加入随机序列DNA库；11点滴定曲线只需要825 fmol的总结合序列。
Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d),(eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in similar to 0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only similar to 825 fmol of total binding sequences were needed for an 11-point titration curve.