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. 2020 Jan 27;59(5):1891-1896.
doi: 10.1002/anie.201912514. Epub 2019 Dec 18.

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells

Mengyi Xiong  1   2 , Zhenglin Yang  3 , Ryan J Lake  2 , Junjie Li  2 , Shanni Hong  2 , Huanhuan Fan  1   2 , Xiao-Bing Zhang  1 , Yi Lu  2   3
Affiliations

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells

Mengyi Xiong et al. Angew Chem Int Ed Engl. .

Abstract

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg2+ -specific 10-23 or Zn2+ -specific 8-17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg2+ or Zn2+ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.

Keywords: DNAzymes; fluorescent proteins; genetically encoded sensors; metal ions.

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Figures

Figure 1.
Figure 1.
(a) The structure of the 10-23 DNAzyme and detection of the RNA substrate cleavage catalysis. (b) Gel electrophoresis analysis of the RNA cleavage efficiency by using 8Dz, 9Dz, 10Dz, 11Dz, and 12Dz with a DNAzyme: substrate ratio of 10:1. (c) The quantification of the gel electrophoresis analysis.
Figure 2.
Figure 2.
Confocal imaging of the cells expressing CloverFP (green channel) and RubyFP (red channel). The cells were transfected with plasmids only (first row) or with plasmids and Dz10TCloverFP with different concentrations of added Mg2+ (second to fourth rows). Scale bar = 100 μm.
Figure 3.
Figure 3.
Confocal imaging of cells co-transfected with CloverFP plasmid (green channel) and Cy5-DNA (red channel) at 6 h and 24h. The lysosomes of cells were stained with Lysotracker Green. (The CloverFP expressing cells exhibited green fluorescence in the whole cells). Scale bar = 20 μm.
Figure 4.
Figure 4.
(a) The contour charts of flow cytometry when Dz10TCloverFP or Dz10HCloverFP was applied to different concentrations of Mg2+. (b) Fluorescent signal ratio of RubyFP to CloverFP in different concentrations of Mg2+ when Dz10TCloverFP or Dz10HCloverFP was used. (c) Comparison of the ratio generated by Dz10TCloverFP or Dz10HCloverFP and Dz10TMCloverFP or Dz10HMCloverFP in the absence or presence of Mg2+.
Scheme 1.
Scheme 1.
Schematic illustration of DNAzyme-mediated genetically encoded sensors for ratiometric imaging of metal ions in living cells.
See this image and copyright information in PMC

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