基于适配体的羧酸酯酶2A生化传感器的设计及应用研究

引用本文: 陈悦, 关晓青, 王亦男, 何青青, 王丹丹, 邹立伟, 汤庆丰, 葛广波. 基于适配体的羧酸酯酶2A生化传感器的设计及应用研究[J]. 分析化学, 2020, 48(11): 1467-1476. doi: 10.19756/j.issn.0253-3820.201283 shu

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

基于适配体的羧酸酯酶2A生化传感器的设计及应用研究

陈悦 ^1, ,
关晓青 ^1, ,
王亦男 ^1, ,
何青青 ^1, ,
王丹丹 ^1, ,
邹立伟 ^1, ,
汤庆丰 ^2, ,
葛广波 ^{1,
,}

1.
上海中医药大学交叉科学研究院, 上海 201203;
2.
上海中医药大学附属普陀医院, 上海 200062

通讯作者: 葛广波,geguangbo@dicp.ac.cn

基金项目:
本文系国家重点研发计划项目（No.2017YFC1700202）、国家自然科学基金项目（Nos.81773687，81922070，81903576）和上海市普陀区卫生系统自主创新科研项目（No.ptkwws201802）资助

摘要: 基于RNA适配体与3，5-二氟-4-羟基亚苄基咪唑啉酮（DFHBI）复合物的荧光特性，结合羧酸酯酶2A（CES2A）的底物偏好性，采用计算机虚拟筛选和实验相结合的策略设计，研发了一种CES2A新型生化传感器。首先，以DFHBI为母核，借助计算机虚拟筛选技术预测了系列DFHBI羧酸酯类衍生物作为CES2A候选底物的潜力。其次，对排名前三位的潜在底物进行合成，并对其化学稳定性和特异性进行了评价，发现3，5-二氟-4-羟基亚苄基咪唑啉酮苯甲酸酯（Ph-DFHBI）对目标酶CES2A显示出极高的特异性和灵敏度。进一步借助同工酶水解实验发现，Ph-DFHBI仅可被CES2A特异性水解，并释放出DFHBI，而人体分布的其它高丰度水解酶（羧酸酯酶1A（CES1A）、乙酰胆碱酯酶（BchE）、丁酰胆碱酯酶（AchE）等）难以水解Ph-DFHBI。进一步在重组单酶和肝组织制备物中考察了CES2A-水解Ph-DFHBI的酶促反应动力学。在生理条件（pH 7.4，37℃）下，CES2A介导的Ph-DFHBI水解反应显示出极高的亲和力和较快的反应速率，K_m=0.66 μmol/L，V_max=18.92 nmol/（min·mg）。在最佳反应条件下，基于Ph-DFHBI的传感器检测CES2A的检出限为14.5 ng/mL。将此生化传感器应用于组织微粒体等复杂生物体系中CES2A活性的快速检测，以及CES2A抑制剂的高通量筛选与评价，均显示出良好的实用性。本研究设计了一种CES2A新型生化传感器，为探究复杂生物体系中CES2A的生物学功能及CES2A调控剂的高效发现提供了有力工具。

关键词:

English

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).

Abstract: 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.

Key words:

Aptamer
/ 3,5-Difluoro-4-hydroxybenzylidene imidazolinone
/ Fluorescent biochemical sensor
/ Carboxylesterase 2A
/ High-throughput screening

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

基于适配体的羧酸酯酶2A生化传感器的设计及应用研究

通讯作者: 葛广波,geguangbo@dicp.ac.cn

English

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

Corresponding author: GE Guang-Bo, geguangbo@dicp.ac.cn

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于适配体的羧酸酯酶2A生化传感器的设计及应用研究

通讯作者: 葛广波,geguangbo@dicp.ac.cn

English

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

Corresponding author: GE Guang-Bo, geguangbo@dicp.ac.cn

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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