Double displacement: An improved bioorthogonal reaction strategy for templated nucleic acid detection.
Double displacement: An improved bioorthogonal reaction strategy for templated nucleic acid detection.
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DOI：
10.1021/bc100165h
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发表时间：
2010-06-16
影响因子：
4.7
通讯作者：
Kool, Eric T.
中科院分区：
文献类型：
--
作者：
Kleinbaum, Daniel J.;Miller, Gregory P.;Kool, Eric T.
淬灭的自配基探针以前被用于靶标模板化的非酶连接策略，用于通过荧光检测细胞中的核酸。在这类探针中，背景信号的一个常见来源是与水和其他细胞亲核试剂的不希望发生的反应。在这里，我们描述了一类新的自连接探针，双位移(DD)探针，它依靠两个置换反应来完全消除附近的一个荧光团。合成了三个潜在的双位移结构，它们都具有两个荧光猝灭/离开基团(氨苯磺酸盐基团)，并在体外和完整的细菌细胞中评价了其与亲核(硫代磷酸)探针的模板反应。所有三种DD探头设计都比单一的Dabsyl控制提供了更好的初始猝灭。在体外等温模板反应中，双位移探针产生的背景信号比以前的单位移探针低得多；对其机理的研究表明，其中一个Dabsyate充当牺牲离开基团，与水发生非特异性反应，但由于另一个猝灭剂基团仍然存在，所以产生的信号很少。需要与特定亲核探针进行模板化反应才能激活信号。双位移式探头提供了大约80倍的开启信号，并且在信号/背景方面比单一的Dabsyl探头提高了2-4倍。在体外双色、基于FRET的双等位基因识别系统中展示了性能最好的探针体系，并被证明能够在核糖体RNA靶点区分两种不同的密切相关细菌。
Quenched autoligation probes have been employed previously in a target-templated nonenzymatic ligation strategy for detecting nucleic acids in cells by fluorescence. A common source of background signal in such probes is undesired reaction with water and other cellular nucleophiles. Here we describe a new class of self-ligating probes, double displacement (DD) probes, that rely on two displacement reactions to fully unquench a nearby fluorophore. Three potential double displacement architectures, all possessing two fluorescence quencher/leaving groups (dabsylate groups), were synthesized and evaluated for templated reaction with nucleophile (phosphorothioate) probes both in vitro and in intact bacterial cells. All three DD probe designs provided substantially better initial quenching than a single-Dabsyl control. In isothermal templated reactions in vitro, double displacement probes yielded considerably lower background signal than previous single-displacement probes; investigation into the mechanism revealed that one dabsylate acts as a sacrificial leaving group, reacting nonspecifically with water, but yielding little signal because another quencher group remains. Templated reaction with the specific nucleophile probe is required to activate a signal. The double displacement probes provided a ca. 80-fold turn-on signal and yielded a 2-4-fold improvement in signal/background over single-Dabsyl probes. The best-performing probe architecture was demonstrated in a two-color, FRET-based two-allele discrimination system in vitro, and was shown to be capable of discriminating between two closely related species of bacteria differing by a single nucleotide at a ribosomal RNA target site.
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影响因子：
3.2
作者：
Franzini, Raphael M.;Kool, Eric T.
通讯作者：
Kool, Eric T.
影响因子：
15
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Jacobsen, Mikkel F.;Clo, Emiliano;Gothelf, Kurt V.
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作者：
Franzini, Raphael M.;Kool, Eric T.
通讯作者：
Kool, Eric T.