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

Abstract

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.

CLC Number:

S811.3

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

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