基于RNA适配体检测体外酶促合成的c-di-GMP和cGAMP  

Detection of invitroEnzyme-synthesized c-di-GMP and cGAMP by Using RNA Aptamer

作　　者：朱彦策 孔江南[1] 陈慧心 钟凯[1] 张超[1] 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)

机构地区：[1]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

出　　处：《畜牧兽医学报》2018年第10期2145-2153,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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)

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、VC2GEMM-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T7RNA聚合酶体外转录体系制备VC2、VC2/g20aRNA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VC2RNA适配体和VC2/g20a RNA适配体,在125mmol·L^(-1 )KCl和30mmol·L^(-1 )MgCl_2溶液中孵育180min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5nmol·L^(-1)和20nmol·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。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 MgCl 2 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.

关 键 词：RNA适配体 C-DI-GMP cGAMP 荧光检测 

分 类 号：S811.3[农业科学—畜牧学]