Citation: ZHANG Yuan-Yuan, CHENG Hui, SUN Yan, WANG Jin-E, WU Zheng-Yan, PEI Ren-Jun. Engineering of Thiamin Pyrophosphate Fluorescent Biosensors Based on Ribozyme Switches in Mammalian Cells[J]. Chinese Journal of Analytical Chemistry, ;2017, 45(2): 157-162. doi: 10.11895/j.issn.0253-3820.160688 shu

Engineering of Thiamin Pyrophosphate Fluorescent Biosensors Based on Ribozyme Switches in Mammalian Cells

ZHANG Yuan-Yuan ^1,2,3, ,
CHENG Hui ³ ,
SUN Yan ³ ,
WANG Jin-E ³ ,
WU Zheng-Yan ² ,
PEI Ren-Jun ³

1.
School of Life Science, Anhui Medical University, Hefei 230032, China;
2.
Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China;
3.
Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China

Received Date: 13 September 2016
Revised Date: 16 November 2016

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos. 21628502, 21507156, 31300096) and the Natural Science Foundation of Jiangsu Province (BK20130357, BK20130351)

Thiamin pyrophosphate (TPP) is a thiamine (vitamin B1) derivative and an essential cofactor in oxidative metabolism of the sugars, fatty acids and amino acids in living cells. By now, numerous TPP-dependent artificial riboswitch systems have been developed to regulate target gene expression but limited in bacteria, fungi or plant cells. Herein, the activating (switch-on) and inhibiting (switch-off) TPP-depended hammerhead ribozyme switches, which are from previous reported structures of prokaryotes screening, were investigated in mammalian cells. These ribozyme switches were inserted into the 3'UTR of the enhanced green fluorescence protein (EGFP) gene to construct the efficient ribozyme-based artificial switches through overlap extension PCR cloning. The HEK293 cells were transfected with the engineered ribozyme switches at increasing concentration of TPP. The EGFP gene-regulatory ability was analyzed with fluorescent microscope and flow cytometry. These TPP-inducible gene regulation devices showed the obvious ligand dose-dependency and excellent specificity. Two "switch-on" and one "switch-off" constructs demonstrated 3.1-fold or 1.9-fold increment and 2.3-fold reduction of EGFP level respectively with 150 μmol/L TPP. The ligand-responsive ribozyme switches, by tuning the change of TPP concentration into the visual reporter genetic expression in cells, enable an efficient development of label-free, noninvasive and high-specific biosensors in living mammalian cells.
- Thiamine pyrophosphate ribozyme switches,
- Enhanced green fluorescence protein,
- Gene expression regulation,
- Fluorescent biosensors,
- Mammalian cells
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