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首页> 外文期刊>Biochemistry >RNA aptamers to S-adenosylhomocysteine: kinetic properties, divalent cation dependency, and comparison with anti-S-adenosylhomocysteine antibody.
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RNA aptamers to S-adenosylhomocysteine: kinetic properties, divalent cation dependency, and comparison with anti-S-adenosylhomocysteine antibody.

机译：RNA对S-腺苷同型半胱氨酸的适体：动力学性质，二价阳离子依赖性以及与抗S-腺苷同型半胱氨酸抗体的比较。

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摘要

To explore the potential of RNA aptamers as small-molecule discriminating devices, we have characterized the properties of aptamers selected from a library of approximately 10(14) variants through their interaction with S-adenosylhomocysteine (SAH, AdoHcy). Competition studies with SAH and azaSAM analogues revealed that the Hoogsteen face of adenine is the main contributor to binding, whereas specificity for SAH is conferred by a secondary contact point at or near the sulfur/thioether of homocysteine (Hcy). Binding specificities were determined by both affinity chromatography and a novel method designed for the biosensor. The kinetic properties of individual aptamers, including the "classic" ATP aptamer that also emerged in our selection, were studied by biosensor analysis. Association rates were slow, but the complexes were stable, suggesting micro- to submicromolar affinities. A solution affinity of approximately 0.1 microM was found for the strongest binding variant under the conditions used for selection (5 mM Mg(2+)). Systematic studies of the effect of Mg(2+) and Mn(2+) on binding, however, revealed that the affinity of the aptamers could be substantially improved, and at optimized conditions of Mn(2+) the affinity of one of the aptamers approached that of an anti-SAH antibody with similar/identical binding specificity. Comparisons with the MAb suggest that the on rate is the limiting factor for high-affinity binding by these aptamers, and comparison with a truncated aptamer shows that shortening of RNA constructs may alter binding kinetics as well as sensitivity to ions.
机译：为了探索RNA适体作为小分子区分装置的潜力，我们通过与S-腺苷同型半胱氨酸（SAH，AdoHcy）的相互作用，对选自约10（14）个变异体的适体的特性进行了表征。 SAH和azaSAM类似物的竞争研究表明，腺嘌呤的Hoogsteen面是结合的主要因素，而SAH的特异性是由高半胱氨酸（Hcy）的硫/硫醚处或附近的次要接触点赋予的。结合特异性通过亲和色谱法和为生物传感器设计的新方法来确定。通过生物传感器分析研究了单个适体的动力学性质，包括也在我们的选择中出现的“经典” ATP适体。缔合速率慢，但复合物稳定，表明微摩尔至亚微摩尔亲和力。在用于选择的条件下（5 mM Mg（2+）），发现最强的结合变体的溶液亲和力约为0.1 microM。系统的研究Mg（2+）和Mn（2+）对结合的影响，但是，发现适体的亲和力可以大大提高，并且在Mn（2+）的优化条件下，其中一个的亲和力适体接近具有相似/相同结合特异性的抗SAH抗体的适体。与MAb的比较表明，打开速率是这些适体高亲和力结合的限制因素，与截短的适体比较表明，RNA构建体的缩短可能会改变结合动力学以及对离子的敏感性。

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