RNA Aptamer-based Biosensors For Detection Of Enzyme-synthesized C-di-GMP And CGAMP In Vitro

Cyclic diadenosine(c-di-AMP),cyclic diguanylic acid(c-di-GMP)and cyclic adenyl-guanylic acid(cGAMP)act as second messenger molecules involved in many aspects of bacterial physiology activities.For example: biofilm formation,differentiation,motility,virulence and the formation of extracellular polysaccharides.It has been reported that c-di GMP transfected into mouse and human cells can stimulate the production of my type interferon through the TBK-IRF3 signaling pathway,indicating its potential as an immunoadjuvant.DncV(VC0179)in V.cholerae is a cyclic dinucleotide cyclase that catalyzes the synthesis of c-di-AMP,c-di-GMP,and cGAMP using ATP and GTP as substrates.In order to investigate the catalytic function of VC0179 as well as the production of c-di-GMP and cGAMP by in vitro enzymatic synthesis,the ability to detect c-di-GMP and cGAMP must first be possible.To date there some in vitro methods of detecting c-di-GMP and cGAMP have been developed.These include high-performance liquid chromatography-coupled tandem mass spectrometry(HPLC-MS/MS),high-performance liquid chromatography(HPLC),fluorescence resonance energy transfer(FRET),fluorescent intercalation,and thin-layer chromatography(TLC).Although some of these methods have been shown to be effective,they are not without their own disadvantages.An RNA aptamer is a single-stranded nucleic acid fragment having a specific complex three-dimensional structure composed of a DNA or ribonucleic acid sequence and having a high affinity for a corresponding target ligand and having a highly specific and stable binding ability.Most recently,RNA aptamer-based biosensors have become a universal tool capable of detecting small molecules both in vitro and in vivo.Samie R.Jaffrey et al.discovered a RNA-based aptamer named spinach,which produces bright green fluorescence once binding to3,5-difluoro-4-hydroxybenzylidene imidazolinone(DFHBI).To date,many RNA aptamer-based biosensors have been developed by conjugating the spinach module with other ligand-binding modules.Colleen A.Kellenberger et al.combined nativeGEMM-I riboswitches with spinach aptamers and constructed aptamers for intracellular c-di-GMP and cGAMP detection.In order to detect in vitro enzymatically synthesized c-di-GMP and cGAMP,this experiment connected the VC2 GEMM-I riboswitch and its mutant VC2/g20 a to spinach2 and t RNA scaffolds,and developed an RNA-based nucleic acid adaptor ligand.Spinach2 has higher thermal stability and folding efficiency than spinach.The constructed RNA aptamers can selectively bind to c-di-GMP and cGAMP.By changing the conditions of RNA aptamers to bind c-di-GMP and cGAMP,RNA aptamers were detected for c-di-GMP and cGAMP optimization conditions.Under optimized detection conditions,the RNA aptamers had an EC50 of 39 n M and 309 n M for c-di-GMP and cGAMP,respectively.In addition,the detection limits of c-di-GMP and cGAMP for VC2 and VC2/g20 a aptamers were 5 n M and 20 n M,respectively.Further experiments revealed that the post-transcriptionally unpurified RNA mixture was capable of significantly detecting in vitro VC0179 synthesized c-di-GMP and cGAMP.the VC2 biosensor was found to bind c-di-GMP selectively and with an affinity(EC50 = 39 n M),while the VC2/g20 a biosensor bound selectively to cGAMP with an affinity(EC50 = 309 n M).In addition,both VC2 and VC2/g20 a sensor possessed a detection limit of 5 n M and 20 n M respectively.Moreover,Further experiments revealed that the post-transcriptionally unpurified RNA mixture was capable of significantly detecting in vitro VC0179 synthesized c-di-GMP and cGAMP.Therefore,the method of detecting c-di-GMP and cGAMP by RNA aptamers used in this experiment is a simpler and faster method for detecting in vitro enzymatically synthesized c-di-GMP and cGAMP.This simple,rapid method can be used to detect other similar cyclic dinucleotide synthetase activities and provides the possibility of detecting c-di-GMP and cGAMP under other conditions.