TokenRNA: a new type of sequence-specific, label-free fluorescent biosensor for folded RNA molecules.
TokenRNA: a new type of sequence-specific, label-free fluorescent biosensor for folded RNA molecules.
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DOI：
10.1002/cbic.200800183
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发表时间：
2008-08-11
期刊：
影响因子：
3.2
通讯作者：
Leontis, Neocles B.
中科院分区：
文献类型：
--
作者：
Afonin, Kirill A.;Danilov, Evgeny O.;Novikova, Irina V.;Leontis, Neocles B.
荧光报告分子是生物学研究中高度灵敏、无干扰和方便的探针。目前用于识别特定核酸序列的技术通常需要与化学修饰的探针互补杂交。[1]最佳的荧光生物传感器应该以高选择性和高对比度快速发出特定分析物存在的信号。最近，已经报道了许多无标记的荧光生物指示剂，包括基于适体的核糖开关作为辅因子的传感器，[2]基于适体的蛋白质传感器，[3]和嵌合适体，其中结合靶标的识别结构域与结合荧光团的适体偶联，使得靶标的存在增加生物指示剂对荧光团的亲和力。[4，5]当荧光团在其结合态和自由态的发射产率不同时，这就提供了一种具有非扰动光学识别的二元传感器。人们希望在其天然环境中检测大分子分析物（例如，结构化RNA分子），而不必使它们变性或展开。在这项工作中，我们展示了一种技术，灵敏，无标记，实时序列特异性识别的预折叠RNA序列。我们使用RNA构建体，其被设计为在可编程的平行结合到特定的预折叠分析物RNA序列后形成荧光团的适体口袋。我们称这些结构为paranemic“token RNA”。
Fluorescent reporters are highly sensitive, nonperturbing, and convenient probes for biological studies. Current techniques for recognition of specific nucleic acid sequences usually require complementary hybridization to chemically modified probes.[1] Optimal fluorescent biosensors should rapidly signal the presence of a specific analyte with high selectivity and high contrast. Recently, a number of label-free, fluorescent bio-indicators have been reported including aptamer-based riboswitches as sensors for cofactors,[2] aptamer-based protein sensors,[3] and chimeric aptamers, in which the recognition domain binding the target was coupled to an aptamer that binds a fluorophore in such a way that the presence of the target increases the affinity of the bioindicator for the fluorophore.[4, 5] When the emission yields of the fluorophore in its bound and free states differ, this provides a binary sensor with nonperturbing optical recognition.It is desirable to detect macromolecular analytes (for example, structured RNA molecules) in their native environment without having to denature or unfold them. In this work, we demonstrate a technique for sensitive, label-free, real-time sequence-specific recognition of prefolded RNA sequences. We use RNA constructs designed to form an aptameric pocket for the fluorophore upon programmable paranemic binding to a specific prefolded analyte RNA sequence. We call these constructs paranemic “token RNAs”.
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