Reduction-Triggered Fluorescent Amplification Probe for the Detection of Endogenous RNAs in Living Human Cells

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• 作者：Kazuhiro Furukawa ; Hiroshi Abe ; Kayo Hibino ; Yasushi Sako ; Satoshi Tsuneda ; Yoshihiro Ito
• 刊名：Bioconjugate Chemistry
• 出版年：2009
• 出版时间：May 20, 2009
• 年：2009
• 卷：20
• 期：5
• 页码：1026-1036
• 全文大小：374K
• 年卷期：v.20,no.5(May 20, 2009)
• ISSN：1520-4812

文摘

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 β-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.