Reduction-triggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells

Bioconjug Chem. 2009 May 20;20(5):1026-36. doi: 10.1021/bc900040t.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azides / chemistry
  • Cell Survival
  • Coumarins / chemistry
  • DNA / chemistry
  • Drug Design
  • Flow Cytometry
  • Fluorescein / chemical synthesis
  • Fluorescein / chemistry
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • HL-60 Cells
  • Humans
  • Kinetics
  • Microscopy, Fluorescence
  • Molecular Imaging / methods*
  • Oxidation-Reduction
  • RNA / metabolism*
  • RNA, Messenger / metabolism
  • RNA, Ribosomal, 28S / metabolism
  • Spectrometry, Fluorescence

Substances

  • Azides
  • Coumarins
  • Fluorescent Dyes
  • RNA, Messenger
  • RNA, Ribosomal, 28S
  • RNA
  • DNA
  • coumarin
  • Fluorescein