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Reduction-triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells.
- Source :
-
Bioconjugate chemistry [Bioconjug Chem] 2009 May 20; Vol. 20 (5), pp. 1026-36. - Publication Year :
- 2009
-
Abstract
- 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 beta-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.
- Subjects :
- Azides chemistry
Cell Survival
Coumarins chemistry
DNA chemistry
Drug Design
Flow Cytometry
Fluorescein chemical synthesis
Fluorescein chemistry
Fluorescent Dyes chemical synthesis
HL-60 Cells
Humans
Kinetics
Microscopy, Fluorescence
Oxidation-Reduction
RNA, Messenger metabolism
RNA, Ribosomal, 28S metabolism
Spectrometry, Fluorescence
Fluorescent Dyes chemistry
Molecular Imaging methods
RNA metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4812
- Volume :
- 20
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Bioconjugate chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 19374406
- Full Text :
- https://doi.org/10.1021/bc900040t