Reduction-Triggered Fluorescent Amplification Probe for the Detection of Endogenous RNAs in Living Human Cells
Reduction-Triggered Fluorescent Amplification Probe for the Detection of Endogenous RNAs in Living Human Cells
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DOI：
10.1021/bc900040t
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发表时间：
2009-05-01
影响因子：
4.7
通讯作者：
Ito, Yoshihiro
中科院分区：
文献类型：
--
作者：
Furukawa, Kazuhiro;Abe, Hiroshi;Ito, Yoshihiro
寡核苷酸模板反应作为活细胞中 RNA 检测的方法引起了人们的关注。此前，已报道了一种还原触发荧光探针，其基于叠氮化物还原来开启荧光。在本文中，我们报告了一种更先进的探针，一种还原触发的荧光放大探针，能够放大目标信号。新合成叠氮甲基荧光素并将其引入探针中。探针上的叠氮基掩蔽荧光素在寡核苷酸模板施陶丁格还原时显示出强烈的开启荧光信号。探针的催化反应在 4 小时内提供了 50 的荧光读数。最后，利用链球菌溶血素O成孔肽将探针引入人白血病HL-60细胞中。我们通过流式细胞术成功检测并定量了背景上方的 28S rRNA 和 P-肌动蛋白 mRNA 信号。此外，还通过荧光显微镜对相同的 RNA 靶标进行了成像。数据表明，还原触发的扩增探针可能是分析活体真核细胞中 RNA 种类的定位、转录或加工的有力工具。
Oligonucleotide-templated reactions are attracting attention as a method for RNA detection in living cells. Previously, a reduction-triggered fluorescence probe has been reported that is based on azide reduction to switch fluorescence on. In this article, we report a more advanced probe, a reduction-triggered fluorescent amplification probe that is capable of amplifying a target signal. Azidomethyl fluorescein was newly synthesized and introduced into a probe. Azido-masked fluorescein on the probe showed a strong turn-on fluorescence signal upon oligonucleotide-templated Staudinger reduction. The catalytic reaction of the probe offered a turnover number of 50 as fluorescence readout within 4 h. Finally, probes were introduced into human leukemia HL-60 cells by use of streptolysin O pore-forming peptide. We successfully detected and quantitated the 28S rRNA and P-Actin mRNA signal above the background by flow cytometry. In addition, the same RNA targets were imaged by fluorescence microscopy. The data suggest that a reduction-triggered amplification probe may be a powerful tool in analyzing the localization, transcription, or processing of RNA species in living eukaryotic cells.