Reduction-triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells.
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.
Furukawa
Kazuhiro
K
Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, Saitama, Japan.
Abe
Hiroshi
H
Hibino
Kayo
K
Sako
Yasushi
Y
Tsuneda
Satoshi
S
Ito
Yoshihiro
Y
D001386:Azides / Q000737:chemistry; D002470:Cell Survival; D003374:Coumarins / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D015195:Drug Design; D005434:Flow Cytometry; D019793:Fluorescein / Q000138:chemical synthesis / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D010084:Oxidation-Reduction; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001386:Azides; D003374:Coumarins; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D012313:RNA; D004247:DNA; C030123:coumarin; D019793:Fluorescein