基于RNA适配体检测体外酶促合成的c-di-GMP和cGAMP

doi:10.11843/j.issn.0366-6964.2018.10.010

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (10): 2145-2153.doi: 10.11843/j.issn.0366-6964.2018.10.010

基于RNA适配体检测体外酶促合成的c-di-GMP和cGAMP

朱彦策, 孔江南, 陈慧心, 钟凯, 张超*

河南农业大学农业部动物生化与营养重点开放实验室, 郑州 450002

收稿日期:2018-01-29 出版日期:2018-10-23 发布日期:2018-10-23
通讯作者: 张超,讲师,主要从事生物化学与分子生物学相关的教学和科研工作,E-mail:lbandeng@126.com
作者简介:朱彦策(1990-),男,河南邓州人,硕士生,主要从事动物生物技术研究,E-mail:zychenau@163.com
基金资助:
河南省基础与前沿技术研究计划项目（142300410153）；河南农业大学科技创新计划（30600967）；河南省自然科学基金资助项目（132300410002）

Detection of in vitro Enzyme-synthesized c-di-GMP and cGAMP by Using RNA Aptamer

ZHU Yan-ce, KONG Jiang-nan, CHEN Hui-xin, ZHONG Kai, ZHANG Chao*

Key Laboratory of Animal Biochemistry and Nutrition of Ministry of Agriculture, Henan Agricultural University, Zhengzhou 450002, China

Received:2018-01-29 Online:2018-10-23 Published:2018-10-23

摘要/Abstract

摘要：

旨在建立简单、快速的检测体外酶促合成的c-di-GMP和cGAMP的方法。本研究以含有T7启动子、VC2 GEMM-1核糖开关序列或其突变序列VC2/g20a、spinach2和tRNA序列的双链DNA为模板，利用T7 RNA聚合酶体外转录体系制备VC2、VC2/g20a RNA适配体。通过检测绿色荧光强度分析RNA适配体对c-di-GMP和cGAMP的结合能力。结果表明，转录得到的RNA经过加热变性、缓慢退火后得到VC2 RNA适配体和VC2/g20a RNA适配体，在125 mmol·L^-1 KCl和30 mmol·L^-1 MgCl₂溶液中孵育180 min的优化结合条件下，VC2适配体与c-di-GMP特异性结合，VC2/g20a适配体与cGAMP特异性结合；c-di-GMP对VC2适配体的半数效应浓度（EC50）为（89±1.7）nmol·L^-1，cGAMP对VC2/g20a适配体的半数效应浓度为（309±4.5）nmol·L^-1；VC2和VC2/g20a能够分别检测低至5 nmol·L^-1和20 nmol·L^-1的c-di-GMP和cGAMP；并且发现转录的RNA混合物在未纯化的情况下同样能够检测VC0179体外酶促合成的c-di-GMP和cGAMP。这种方法可以简单快速的检测VC0179体外酶促合成的c-di-GMP和cGAMP，并且为研究其他c-di-GMP和cGAMP合成酶活性提供潜在可能。

Abstract:

The aim of this study was to construct the method to simply and robustly detect the in vitro enzyme-synthesized c-di-GMP and cGAMP. In this study, the double-stranded DNA containing T7 promoter, VC2 GEMM-I ribose switch sequence or VC2/g20a, spinach2 and tRNA sequence was as the template, T7 RNA polymerase in vitro transcription system was used to prepare VC2, VC2/g20a RNA aptamers. In addition, the binding abilities of RNA aptamer to c-di-GMP and cGAMP were determined by examining the green fluorescence intensity. The results showed that the VC2 RNA aptamer and VC2/g20a RNA aptamer were obtained through the process in which transcribed RNA was heated and then was cooled slowly. Under the optimized conditions of 125 mmol·L^-1 KCl, 30 mmol·L^-1 MgCl₂ for 180 min, VC2 and VC2/g20a aptamers could bind to c-di-GMP and cGAMP, respectively, both with specificity and selectivity. In addition, the half effect concentration (EC50) of c-di-GMP for VC2 aptamer was (89±1.7) nmol·L^-1, the EC50 of cGAMP for VC2/g20a aptamer was (309±4.5) nmol·L^-1. The detection limit of VC2 and VC2/g20a aptamers for c-di-GMP and cGAMP were 5 nmol·L^-1 and 20 nmol·L^-1, respectively. Moreover, it was shown that the transcribed RNA mixture without purification could noticeably detect c-di-GMP and cGAMP by VC0179 synthesized. Thus, this simple and robust method could be useful in detecting the c-di-GMP and cGAMP by VC0179 synthesized in vitro,and which might be applied to detect the activity of other c-di-GMP and cGAMP synthases.

中图分类号:

S811.3

朱彦策, 孔江南, 陈慧心, 钟凯, 张超. 基于RNA适配体检测体外酶促合成的c-di-GMP和cGAMP[J]. 畜牧兽医学报, 2018, 49(10): 2145-2153.

ZHU Yan-ce, KONG Jiang-nan, CHEN Hui-xin, ZHONG Kai, ZHANG Chao. Detection of in vitro Enzyme-synthesized c-di-GMP and cGAMP by Using RNA Aptamer[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(10): 2145-2153.

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基于RNA适配体检测体外酶促合成的c-di-GMP和cGAMP

Detection of in vitro Enzyme-synthesized c-di-GMP and cGAMP by Using RNA Aptamer

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