Smart aptamers facilitate multi-probe affinity analysis of proteins with ultra-wide dynamic range of measured concentrations
Smart aptamers facilitate multi-probe affinity analysis of proteins with ultra-wide dynamic range of measured concentrations
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DOI：
10.1021/ja072269p
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发表时间：
2007-06-13
影响因子：
15
通讯作者：
Krylov, Sergey N.
中科院分区：
文献类型：
--
作者：
Drabovich, Andrei P.;Okhonin, Victor;Krylov, Sergey N.
在许多生理、病理和生物技术过程中，蛋白质浓度可以在几个数量级上变化。对这些过程的研究需要对具有宽动态范围的精确测量浓度的蛋白质进行亲和分析。宽的动态范围可以用多种亲和探针实现，所述多种亲和探针以显著不同的平衡常数（K-d）结合靶标。在这种多探针亲和分析中的每个探针负责检测在其K-d值附近的浓度范围内的靶标。由于缺乏具有宽范围Kd和高选择性的通用亲和探针，迄今为止蛋白质的多探针亲和分析尚未变得实用。动力学毛细管电泳（KCE）是近年来发展起来的一种高效的DNA适体分离技术，具有广泛的选择性和选择性。在这里，我们证明，第一次，这样的适体可以促进多探针亲和分析的蛋白质与超宽的动态范围内的测量浓度。我们的结果表明，三适体分析的浓度动态范围超过4个数量级。据我们所知，这是有史以来报道的蛋白质亲和力分析的最宽动态范围。有利地，多适体分析中的蛋白质浓度可以使用简单的免校准方法来确定。这项工作证明了智能适体的广泛的预定义结合参数可以为定量亲和力分析带来新的能力。智能适体的相同特征使得它们在细胞内过程的研究中成为潜在的不可或缺的分子工具。
Protein concentration can vary over several orders of magnitude in many physiological, pathological, and biotechnological processes. Studies of these processes require affinity analysis of proteins with a wide dynamic range of accurately measured concentrations. The wide dynamic range can be achieved with multiple affinity probes that bind the target with significantly different equilibrium constants (K-d). Every probe in such a multi-probe affinity analysis is responsible for detection of the target in a range of concentrations around its K-d value. A multi-probe affinity analysis of proteins has not become practical so far due to the lack of generic affinity probes with a wide range of K-d and high selectivity. Kinetic capillary electrophoresis (KCE) has been recently proven to generate smart DNA aptamers with a wide range of predefined values of K-d and high selectivity. Here, we demonstrate, for the first time, that such aptamers can facilitate multi-probe affinity analysis of a protein with an ultra-wide dynamic range of measured concentrations. Our results showed that a three-aptamer analysis had a concentration dynamic range of more than 4 orders of magnitude. To the best of our knowledge, this is the widest dynamic range ever reported for affinity analyses of proteins. Advantageously, protein concentration in a multi-aptamers analysis can be determined using a simple calibration-free approach. This work proves that the wide range of predefined binding parameters of smart aptamers can bring new capabilities to quantitative affinity analyses. The same feature of smart aptamers makes them potentially indispensable molecular tools in studies of intracellular processes.