Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation.
Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation.
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通过核酸酶定向截断将结构转换功能引入小分子结合适体
DOI：
10.1093/nar/gky305
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发表时间：
2018-07-27
影响因子：
14.9
通讯作者：
Xiao Y
中科院分区：
文献类型：
--
作者：
Wang Z;Yu H;Canoura J;Liu Y;Alkhamis O;Fu F;Xiao Y
摘要我们报道了一种广泛适用的酶消化策略，用于将结构转换功能引入小分子结合适体。该程序基于我们的发现，即核酸外切酶III（Exo III）对适体的消化被靶标结合极大地抑制。作为示范，我们进行Exo III消化的预折叠的三通连接（TWJ）结构的可卡因结合适体和茎环结构的ATP结合适体。在没有靶标的情况下，Exo III催化两种适体的3′至5′消化，形成短的单链产物。在添加靶标后，Exo III消化在靶标结合结构域之前四个碱基停止，形成主要的靶标结合适体消化产物。我们证明了靶标结合对于Exo III抑制至关重要。然后，我们确定两种适体的所得消化产物表现出亲本适体中不存在的靶诱导的结构转换功能，同时仍保持高的靶结合亲和力。我们确认，这些截短的适体具有这种功能，通过使用核酸外切酶I为基础的消化试验，并进一步评估电化学适体为基础的可卡因传感器和荧光猝灭剂ATP测定这一特点。我们相信我们的Exo III消化方法应该适用于从其他含TWJ或茎环的小分子结合适体产生结构转换适体，大大简化了基于折叠的适体传感器的功能化传感器元件的产生。
Abstract We report a broadly applicable enzyme digestion strategy for introducing structure-switching functionality into small-molecule-binding aptamers. This procedure is based on our discovery that exonuclease III (Exo III) digestion of aptamers is greatly inhibited by target binding. As a demonstration, we perform Exo III digestion of a pre-folded three-way-junction (TWJ)-structured cocaine-binding aptamer and a stem–loop-structured ATP-binding aptamer. In the absence of target, Exo III catalyzes 3′-to-5′ digestion of both aptamers to form short, single-stranded products. Upon addition of target, Exo III digestion is halted four bases prior to the target-binding domain, forming a major target-bound aptamer digestion product. We demonstrated that target-binding is crucial for Exo III inhibition. We then determine that the resulting digestion products of both aptamers exhibit a target-induced structure-switching functionality that is absent in the parent aptamer, while still retaining high target-binding affinity. We confirm that these truncated aptamers have this functionality by using an exonuclease I-based digestion assay and further evaluate this characteristic in an electrochemical aptamer-based cocaine sensor and a fluorophore-quencher ATP assay. We believe our Exo III-digestion method should be applicable for the generation of structure-switching aptamers from other TWJ- or stem–loop-containing small-molecule-binding aptamers, greatly simplifying the generation of functionalized sensor elements for folding-based aptasensors.
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