Citation: CHEN Yue, GUAN Xiao-Qing, WANG Yi-Nan, HE Qing-Qing, WANG Dan-Dan, ZOU Li-Wei, TANG Qing-Feng, GE Guang-Bo. Development and Application of RNA Aptamer-based Biochemical Sensor for Carboxylesterase 2A[J]. Chinese Journal of Analytical Chemistry, ;2020, 48(11): 1467-1476. doi: 10.19756/j.issn.0253-3820.201283 shu

Development and Application of RNA Aptamer-based Biochemical Sensor for Carboxylesterase 2A

1.
Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;
2.
Putuo Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

Corresponding author: GE Guang-Bo, geguangbo@dicp.ac.cn
Received Date: 18 May 2020
Revised Date: 19 August 2020

Fund Project: This work was supported by the National Key Research and Development Project (Nos. 2017YFC1700202), the National Natural Science Foundation of China (Nos. 81773687, 81922070, 81903576), and the Health System Independent Innovation Science Foundation of Shanghai Putuo District (No. ptkwws201802).

Based on the fluorescent characteristics of 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and RNA aptamer complex, a novel biochemical sensor was designed for sensing carboxylesterase 2A (CES2A) activity via combination of virtual screening and experimental validation. Firstly, virtual screening was applied to predict the potential of a series of DFHBI derivatives as candidate substrates for CES2A. After then, the TOP3 candidate substrates were synthesized, while their chemical stability and specificity towards CES2A were assayed. The results showed that 3,5-difluoro-4-hydroxybenzylidene imidazolinone benzoate (Ph-DFHBI) displayed extremely high specificity and the best response to CES2A. Further investigations demonstrated that Ph-DFHBI could be selectively hydrolyzed by CES2A, while other abundant hydrolases (such as CES1A, BchE and AchE) in the human body were hardly catalyzing Ph-DFHBI hydrolysis. To quantitatively measure CES2A activity in real samples, the enzymatic kinetic behaviors of Ph-DFHBI hydrolysis in both recombinant CES2A and human liver preparations were further investigated. Under physiological conditions (pH 7.4 at 37℃), Ph-DFHBI exhibited extremely high affinity and relatively high turnover rate, with the K_m value of 0.66 μmol/L, and the V_max value of 18.92 nmol/(min·mg). Under the optimal conditions, the limit of detection of Ph-DFHBI for sensing CES2A was 14.5 ng/mL. On the basis of the above mentioned findings, this biochemical sensor was successfully utilized to rapidly measure the CES2A activities in complex biological systems (such as tissue preparations), as well as high-throughput screening of CES2A inhibitors. Collectively, this study developed a novel biochemical sensor for CES2A, which provided a powerful tool for exploring the biological functions of CES2A and for efficient discovery of CES2A modulators in complex biological systems.
- Aptamer,
- 3,5-Difluoro-4-hydroxybenzylidene imidazolinone,
- Fluorescent biochemical sensor,
- Carboxylesterase 2A,
- High-throughput screening
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