Xiong Mengyi, Yang Zhenglin, Lake Ryan J, Li Junjie, Hong Shanni, Fan Huanhuan, Zhang Xiao-Bing, Lu Yi
Molecular Science and Biomedicine Laboratory (MBL), State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University Changsha 410082 (P. R. China).
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (USA).
Angew Chem Weinheim Bergstr Ger. 2020 Jan 27;132(5):1907-1912. doi: 10.1002/ange.201912514. Epub 2019 Nov 20.
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 Mg-specific 10-23 or Zn-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 Mg or Zn 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.
基因编码荧光蛋白（FPs）已被用于金属离子检测。然而，由于缺乏可与FPs融合的金属结合蛋白或肽，以及难以将金属离子的结合转化为荧光信号的变化，它们的应用仅限于少数几种金属离子。我们在此报告使用镁特异性10-23或锌特异性8-17 RNA切割脱氧核酶来调节FPs的表达，作为一类新型的金属离子比率荧光传感器。具体而言，我们证明了利用脱氧核酶通过切割Clover2（绿色荧光蛋白（GFP）的变体）的mRNA来抑制其表达，而红色荧光蛋白（RFP）的突变体Ruby2的表达不受影响。利用共聚焦成像和流式细胞术均可检测HeLa细胞中的镁或锌。由于可以获得多种金属特异性脱氧核酶，该方法可能适用于许多其他金属离子的成像，从而扩大了当前基于基因编码荧光蛋白的传感器的范围。
