Aptamers selected for higher-affinity binding are not more specific for the target ligand
Aptamers selected for higher-affinity binding are not more specific for the target ligand
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DOI：
10.1021/ja060952q
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发表时间：
2006-06-21
影响因子：
15
通讯作者：
Szostak, Jack W.
中科院分区：
文献类型：
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作者：
Carothers, James M.;Oestreich, Stephanie C.;Szostak, Jack W.
先前对11种结合鸟苷三磷酸（GTP）和K(d)s范围从8 μ M到9 nM的体外选择RNA适配体的研究表明，需要更多的信息来确定高亲和力适配体的结构。我们感兴趣的是了解更复杂的适体如何与配体产生更高的亲和力。在体外选择产生的结构性解决方案的功能问题，是尽可能简单的信息内容，需要定义他们。长期以来，人们一直认为提高适体亲和力的最简单方法是增加配体RNA结合袋的形状和官能团互补性。这一论点奠定了一种假设的基础，即选择高亲和力的适体会自动导致与目标分子结合更特异性的结构。在这里，我们通过与16种不同的GTP化学类似物进行竞争结合研究，检验了11种GTP适配体的结合特异性。适配体具有不同的特异性模式，这意味着每个RNA在结构上都是GTP结合问题的独特解决方案。然而，这些实验未能提供证据证明高亲和力适配体更特异性地结合GTP。我们认为改善适体K(d)s最简单的方法可能是通过增加分子内RNA-RNA相互作用来增加RNA三级结构的稳定性；越来越特异的配体结合可能只出现在直接选择特异性的反应中。
Previous study of eleven different in vitro-selected RNA aptamers that bind guanosine triphosphate (GTP) with K(d)s ranging from 8 mu M to 9 nM showed that more information is required to specify the structures of the higher-affinity aptamers. We are interested in understanding how the more complex aptamers achieve higher affinities for the ligand. In vitro selection produces structural solutions to a functional problem that are are as simple as possible in terms of the information content needed to define them. It has long been assumed that the simplest way to improve the affinity of an aptamer is to increase the shape and functional group complementarity of the RNA binding pocket for the ligand. This argument underlies the hypothesis that selection for higher-affinity aptamers automatically leads to structures that bind more specifically to the target molecule. Here, we examined the binding specificities of the eleven GTP aptamers by carrying out competition binding studies with sixteen different chemical analogues of GTP. The aptamers have distinct patterns of specificity, implying that each RNA is a structurally unique solution to the problem of GTP binding. However, these experiments failed to provide evidence that higher-affinity aptamers bind more specifically to GTP. We suggest that the simplest way to improve aptamer K(d)s may be to increase the stability of the RNA tertiary structure with additional intramolecular RNA-RNA interactions; increasingly specific ligand binding may emerge only in response to direct selection for specificity.