Ultrafast dynamics show that the theophylline and 3-methylxanthine aptamers employ a conformational capture mechanism for binding their ligands.
Ultrafast dynamics show that the theophylline and 3-methylxanthine aptamers employ a conformational capture mechanism for binding their ligands.
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DOI：
10.1021/bi100106c
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发表时间：
2010-04-06
期刊：
影响因子：
2.9
通讯作者：
Xia, Tianbing
中科院分区：
文献类型：
--
作者：
Lee, Sang Won;Zhao, Liang;Pardi, Arthur;Xia, Tianbing
RNA通常表现出高度的构象动力学和异质性，导致了崎岖的能量格局。然而，构象异质性和快速动力学在分子识别或RNA功能中的作用还没有被广泛阐明。利用超快时间分辨荧光光谱实验研究了茶碱结合RNA适配子的皮秒动力学。这些研究表明，在游离RNA中存在多种构象，表明该适配子采用构象捕获机制进行配体结合。内部环中残基27的碱基在游离RNA中至少以三种构象状态存在，包括具有指示不同碱基堆积作用的不同荧光衰减特征的结合能力状态和非能力状态。各向异性实验也检测到了皮秒动力学，这些运动表明基数为27的额外动力学。皮秒数据表明，茶碱结合使碱基27的构象平衡从主要堆积在游离RNA中转变为主要未堆积在RNA-茶碱复合体中，如在先前的核磁共振结构中观察到的那样。相反，第二个内部环中的碱基10大部分是在自由RNA中预组织的，与它在G11和G25之间堆积一致，如在结合状态下观察到的那样。在与3-甲基黄嘌呤结合比茶碱亲和力更高的修饰适配子上也测量了皮秒动力学。修饰的适配子在适配子-3-甲基黄嘌呤复合体中的异质性比在茶碱适配子-茶碱复合体中观察到的少。
RNAs often exhibit a high degree of conformational dynamics and heterogeneity, leading to a rugged energy landscape. However, the roles of conformational heterogeneity and rapid dynamics in molecular recognition or RNA function have not been extensively elucidated. Ultrafast time-resolved fluorescence spectroscopic experiments were used here to probe picosecond dynamics of the theophylline-binding RNA aptamer. These studies showed that multiple conformations are populated in the free RNA indicating that this aptamer employs a conformational capture mechanism for ligand binding. The base on residue 27 in an internal loop exists in at least three conformational states in the free RNA, including binding competent and incompetent states that have distinct fluorescence decay signatures indicating different base stacking interactions. Picosecond dynamics were also detected by anisotropy experiments, where these motions indicate additional dynamics for base 27. The picosecond data show that theophylline binding shifts the equilibrium for conformations of base 27 from primarily stacked in the free RNA to mostly unstacked in the RNA-theophylline complex, as observed in the previous NMR structure. In contrast, base 10 in a second internal loop is mostly pre-organized in the free RNA, consistent with it being stacked between G11 and G25, as is observed in the bound state. Picosecond dynamics were also measured on a modified aptamer that binds with higher affinity to 3-methylxanthine than theophylline. The modified aptamer shows less heterogeneity in the aptamer-3-methylxanthine complex than what is observed in the theophylline aptamer-theophylline complex.
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