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

Autor: Shanni Hong, Huanhuan Fan, Yi Lu, Zhenglin Yang, Xiao-Bing Zhang, Jun Jie Li, Ryan J. Lake, Mengyi Xiong
Rok vydání: 2022
Předmět:
Zdroj: Angew Chem Weinheim Bergstr Ger
Angew Chem Int Ed Engl
ISSN: 0044-8249
Popis: Genetically encoded fluorescent proteins have been used for metal ion detections by combining fluorescent proteins with metal-binding proteins or peptides. However, their applications are largely restricted to a limited number of metal ions, such as Ca(2+) and Zn(2+), due to the lack of available metal-binding proteins or peptides that can be fused to fluorescent proteins and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. To overcome these limitations, we report herein the use of Mg(2+)-specific 10-23 or Zn(2+)-specific 8-17 RNA-cleaving DNAzymes to regulate the expression of fluorescent proteins 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 fluorescent protein, by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red fluorescent protein, is not affected. The Mg(2+) or Zn(2+) in Hela cells can be detected using both fluorescent confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes, such as for Mg(2+), Na(+), Cu(2+), Zn(2+), Pb(2+), Hg(2+), Ag(+), and UO(2)(2+), can be obtained through in vitro selection, and the resulting DNAzymes often share a similar secondary structure and reaction mechanism, the method described in this work can likely be applied to imaging many other metal ions and thus significantly expand beyond the range of the current genetically-encoded fluorescent proteins, allowing this class of sensors to be even more powerful in providing deeper understanding of the roles of metal ions in biology.
Databáze: OpenAIRE
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