Experimental and mathematical evidence that thrombin-binding aptamers form a 1 aptamer:2 protein complex
Experimental and mathematical evidence that thrombin-binding aptamers form a 1 aptamer:2 protein complex
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发表时间：
2018-10
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通讯作者：
Kepler S. Mears;Dani Markus;O. Ogunjimi;R. Whelan
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作者：
Kepler S. Mears;Dani Markus;O. Ogunjimi;R. Whelan
凝血酶结合的15聚体和29聚体的单链DNA适配子是一个广泛使用的模型系统。尽管它们无处不在，但关于适配子-蛋白质相互作用的性质仍然存在争议。已报道的亲和力差异很大；金属离子在结合中的作用尚不清楚；络合物的结构存在争议。我们询问了仪器、缓冲液和数学模型对凝血酶适配子与其靶标的表观亲和力的影响。仪器方法对15聚体的亲和常数有显著影响，对29聚体的表观亲和力影响较小。缓冲液成分和离子环境没有显著影响。亲和探针毛细管电泳法从29mer适配子和凝血酶样品中发现了明显的峰，支持1适配子：2蛋白复合体的模型。与高质量数据的五个数学模型的拟合进一步支持这一化学计量学，因为两个适配子的结合符合Hill系数为1的Hill方程。我们的结果表明，仪器方法和数学模型影响凝血酶适配子的表观亲和力，两个适配子通过诱导拟合机制与1适配子：2蛋白质化学计量比结合凝血酶。
The thrombin-binding 15mer and 29mer ssDNA aptamers are a widely used model system. Despite their ubiquity, controversies persist regarding the nature of the aptamer-protein interactions. Reported affinities vary widely; the role of metal ions in binding is unclear; the structure of the complex is contested. We interrogated the effects of instrument, buffer, and mathematical model on apparent affinities of thrombin aptamers for their target. Instrumental method had a pronounced effect on affinity constants for the 15mer and marginal effect the apparent affinity of the 29mer. Buffer composition and ionic environment did not have significant effects. Affinity probe capillary electrophoresis experiments revealed distinct peaks from samples of 29mer aptamer and thrombin, supporting the model of a 1 aptamer:2 protein complex. Fits to high quality data with five mathematical models further support this stoichiometry, as the binding of both aptamers was best described by the Hill equation with Hill coefficients > 1. Our results indicate that the instrumental method and mathematical model influence apparent affinity of thrombin aptamers and that both aptamers bind thrombin in a 1 aptamer: 2 protein stoichiometry through an induced fit mechanism.