RESUMEN
Riboswitches are common gene regulatory units mostly found in bacteria that are capable of altering gene expression in response to a small molecule. These structured RNA elements consist of two modular subunits: an aptamer domain that binds with high specificity and affinity to a target ligand and an expression platform that transduces ligand binding to a gene expression output. Significant progress has been made in engineering novel aptamer domains for new small molecule inducers of gene expression. Modified expression platforms have also been optimized to function when fused with both natural and synthetic aptamer domains. As this field expands, the use of these privileged scaffolds has permitted the development of tools such as RNA-based fluorescent biosensors. In this review, we summarize the methods that have been developed to engineer new riboswitches and highlight applications of natural and synthetic riboswitches in enzyme and strain engineering, in controlling gene expression and cellular physiology, and in real-time imaging of cellular metabolites and signals.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Técnicas Biosensibles/métodos , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética/métodos , Riboswitch , Aptámeros de Nucleótidos/síntesis química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ligandos , Imagen Molecular/métodos , Rhodocyclaceae/genética , Rhodocyclaceae/metabolismoRESUMEN
Tumor necrosis factor-α (TNF-α) is the most potent proinflammatory cytokine in mammals. The degradation of TNF-α mRNA is critical for restricting TNF-α synthesis and involves a constitutive decay element (CDE) in the 3' UTR of the mRNA. Here, we demonstrate that the CDE folds into an RNA stem-loop motif that is specifically recognized by Roquin and Roquin2. Binding of Roquin initiates degradation of TNF-α mRNA and limits TNF-α production in macrophages. Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE sequences are highly conserved and are found in more than 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, CDE-containing mRNAs were identified as the primary targets of Roquin on a transcriptome-wide scale. Thus, Roquin proteins act broadly as mediators of mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs.
Asunto(s)
Macrófagos/metabolismo , Estabilidad del ARN , Proteínas Represoras/metabolismo , Factor de Necrosis Tumoral alfa/genética , Ubiquitina-Proteína Ligasas/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular , Humanos , Inflamación/metabolismo , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Motivos de Nucleótidos , ARN Mensajero/química , Alineación de SecuenciaRESUMEN
Bacterial second messengers are important for regulating diverse bacterial lifestyles. Cyclic di-GMP (c-di-GMP) is produced by diguanylate cyclase enzymes, named GGDEF proteins, which are widespread across bacteria. Recently, hybrid promiscuous (Hypr) GGDEF proteins have been described in some bacteria, which produce both c-di-GMP and a more recently identified bacterial second messenger, 3',3'-cyclic-GMP-AMP (cGAMP). One of these proteins was found in the predatory Bdellovibrio bacteriovorus, Bd0367. The bd0367 GGDEF gene deletion strain was found to enter prey cells, but was incapable of leaving exhausted prey remnants via gliding motility on a solid surface once predator cell division was complete. However, it was unclear which signal regulated this process. We show that cGAMP signalling is active within B. bacteriovorus and that, in addition to producing c-di-GMP and some c-di-AMP, Bd0367 is a primary producer of cGAMP in vivo. Site-directed mutagenesis of serine 214 to an aspartate rendered Bd0367 into primarily a c-di-GMP synthase. B. bacteriovorus strain bd0367S214D phenocopies the bd0367 deletion strain by being unable to glide on a solid surface, leading to an inability of new progeny to exit from prey cells post-replication. Thus, this process is regulated by cGAMP. Deletion of bd0367 was also found to be incompatible with wild-type flagellar biogenesis, as a result of an acquired mutation in flagellin chaperone gene homologue fliS, implicating c-di-GMP in regulation of swimming motility. Thus the single Bd0367 enzyme produces two secondary messengers by action of the same GGDEF domain, the first reported example of a synthase that regulates multiple second messengers in vivo. Unlike roles of these signalling molecules in other bacteria, these signal to two separate motility systems, gliding and flagellar, which are essential for completion of the bacterial predation cycle and prey exit by B. bacteriovorus.
Asunto(s)
Bdellovibrio bacteriovorus , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/genética , Bdellovibrio bacteriovorus/metabolismo , Nucleótidos Cíclicos/metabolismoRESUMEN
Singlet oxygen generation has long been considered the key feature that allows genetically encoded fluorescent tags to produce polymeric contrast agents for electron microscopy. Optimization of the singlet oxygen sensitization quantum yield has not included the effects of electron-rich monomers on the sensitizer's photocycle. We report that at monomer concentrations employed for staining, quenching by electron transfer is the primary deactivation pathway for photoexcitations. A simple photochemical model including contributions from both processes reproduces the observed reaction rates and indicates that most of the product is driven by pathways that involve electron transfer with monomersânot by the sensitization of singlet oxygen. Realizing the importance of these competing reaction pathways offers a new paradigm to guide the development of genetically encodable tags and suggests opportunities to expand the materials scope and growth conditions for polymeric contrast agents (e.g., biocompatible monomers, O2 poor environments).
Asunto(s)
Medios de Contraste , Polimerizacion , Transporte de Electrón , Medios de Contraste/química , Oxígeno Singlete/química , Flavoproteínas/química , Flavoproteínas/metabolismo , Fármacos Fotosensibilizantes/química , Procesos FotoquímicosRESUMEN
Malate is a key intermediate in the citric acid cycle, an enzymatic cascade that is central to cellular energy metabolism and that has been applied to make biofuel cells. To enable real-time sensing of malate levels, we have engineered a genetically encoded, protein-based fluorescent biosensor called Malon specifically responsive to malate by performing structure-based mutagenesis of the Cache-binding domain of the Citron GFP-based biosensor. Malon demonstrates high specificity and fluorescence activation in response to malate, and has been applied to monitor enzymatic reactions in vitro. Furthermore, we successfully incorporated Malon into redox polymer hydrogels and bacterial cells, enabling analysis of malate levels in these materials and living systems. These results show the potential for fluorescent biosensors in enzymatic cascade monitoring within biomaterials and present Malon as a novel tool for bioelectronic devices.
Asunto(s)
Técnicas Biosensibles , Hidrogeles , Malatos , Técnicas Biosensibles/métodos , Hidrogeles/química , Malatos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMEN
The Hypr cGAMP signaling pathway was discovered via the function of the riboswitch. In this study, we show the development of a method for affinity capture followed by sequencing to identify non-coding RNA regions that bind nucleotide signals such as cGAMP. The RNAseq of affinity-captured cGAMP riboswitches from the Geobacter sulfurreducens transcriptome highlights general challenges that remain for this technique. Furthermore, by applying riboswitch reporters in vivo, we identify new growth conditions and transposon mutations that affect cGAMP levels in G. sulfurreducens. This work reveals an extensive regulatory network and supports a second functional cGAMP synthase gene in G. sulfurreducens. The activity of the second synthase was validated using riboswitch-based fluorescent biosensors, and is the first known example of an active enzyme with a variant GGDDF motif.
Asunto(s)
Proteínas Bacterianas/metabolismo , Geobacter/metabolismo , Mutación , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/metabolismo , Riboswitch , Transcriptoma , Proteínas Bacterianas/genética , Geobacter/genética , Nucleotidiltransferasas/genética , Transducción de SeñalRESUMEN
3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.
Asunto(s)
Proteínas Bacterianas/metabolismo , Myxococcus xanthus/enzimología , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismoRESUMEN
Bacteria contain a diverse set of RNAs to provide tight regulation of gene expression in response to environmental stimuli. Bacterial small RNAs (sRNAs) work in conjunction with protein cofactors to bind complementary mRNA sequences in the cell, leading to up- or downregulation of protein synthesis. In vivo imaging of sRNAs can aid in understanding their spatiotemporal dynamics in real time, which inspires new ways to manipulate these systems for a variety of applications including synthetic biology and therapeutics. Current methods for sRNA imaging are quite limited in vivo and do not provide real-time information about fluctuations in sRNA levels. Herein, we describe our efforts toward the development of an RNA-based fluorescent biosensor for bacterial sRNA both in vitro and in vivo. We validated these sensors for three different bacterial sRNAs in Escherichia coli and demonstrated that the designs provide a bright, sequence-specific signal output in response to exogenous and endogenous RNA targets.
Asunto(s)
Técnicas Biosensibles/métodos , Escherichia coli/genética , Colorantes Fluorescentes/química , ARN Bacteriano/análisis , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Sitios de Unión , Proteínas de Escherichia coli/genética , Citometría de Flujo , Proteína de Factor 1 del Huésped/genética , Hibridación de Ácido Nucleico , ARN Bacteriano/química , Espectrometría de FluorescenciaRESUMEN
Cyclic dinucleotides (CDNs) trigger the innate immune response in eukaryotic cells through the stimulator of interferon genes (STING) signaling pathway. To decipher this complex cellular process, a better correlation between structure and downstream function is required. Herein, we report the design and immunostimulatory effect of a novel group of c-di-GMP analogues. By employing an "atomic mutagenesis" strategy, changing one atom at a time, a class of gradually modified CDNs was prepared. These c-di-GMP analogues induce type-I interferon (IFN) production, with some being more potent than c-di-GMP, their native archetype. This study demonstrates that CDN analogues bearing modified nucleobases are able to tune the innate immune response in eukaryotic cells.
Asunto(s)
GMP Cíclico/inmunología , Interferones/inmunología , Nucleótidos Cíclicos/inmunología , GMP Cíclico/análogos & derivados , GMP Cíclico/química , Inmunidad Innata , Interferones/química , Interferones/genética , Nucleótidos Cíclicos/química , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
RNA-based fluorescent (RBF) biosensors have been applied to detect a variety of metabolites in vitro and in live cells. They are designed by combining the ligand sensing domain of natural riboswitches with in vitro selected fluorogenic aptamers. Different biosensor topologies have been developed to accommodate the diversity of riboswitch structures. Here we show that circular permutation of the riboswitch ligand sensing domain also gives functional biosensors, using the SAM-I riboswitch as our model. We reveal that this design can enhance fluorescence turn-on and ligand binding affinity compared to the non-permuted topology.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Conformación de Ácido Nucleico , Riboswitch/genética , S-Adenosilmetionina/química , Aptámeros de Nucleótidos/genética , Fluorescencia , Ligandos , Transcripción GenéticaRESUMEN
Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Nucleótidos Cíclicos/biosíntesis , Liasas de Fósforo-Oxígeno/metabolismo , Deltaproteobacteria/enzimología , Proteínas de Escherichia coli/química , Liasas de Fósforo-Oxígeno/química , Conformación ProteicaRESUMEN
Intracellular signaling enzymes drive critical changes in cellular physiology and gene expression, but their endogenous activities in vivo remain highly challenging to study in real time and for individual cells. Here we show that flow cytometry can be performed in complex media to monitor single-cell population distributions and dynamics of cyclic di-GMP signaling, which controls the bacterial colonization program. These in vivo biochemistry experiments are enabled by our second-generation RNA-based fluorescent (RBF) biosensors, which exhibit high fluorescence turn-on in response to cyclic di-GMP. Specifically, we demonstrate that intracellular levels of cyclic di-GMP in Escherichia coli are repressed with excess zinc, but not with other divalent metals. Furthermore, in both flow cytometry and fluorescence microscopy setups, we monitor the dynamic increase in cellular cyclic di-GMP levels upon zinc depletion and show that this response is due to de-repression of the endogenous diguanylate cyclase DgcZ. In the presence of zinc, cells exhibit enhanced cell motility and increased sensitivity to antibiotics due to inhibited biofilm formation. Taken together, these results showcase the application of RBF biosensors in visualizing single-cell dynamic changes in cyclic di-GMP signaling in direct response to environmental cues such as zinc and highlight our ability to assess whether observed phenotypes are related to specific signaling enzymes and pathways.
Asunto(s)
Técnicas Biosensibles , GMP Cíclico/análogos & derivados , Escherichia coli/metabolismo , ARN/química , Análisis de la Célula Individual , Zinc/metabolismo , GMP Cíclico/metabolismo , Citometría de Flujo , Microscopía Fluorescente , Transducción de SeñalRESUMEN
Bacteria occupy a diverse set of environmental niches with differing oxygen availability. Anaerobic environments such as mammalian digestive tracts and industrial reactors harbor an abundance of both obligate and facultative anaerobes, many of which play significant roles in human health and biomanufacturing. Studying bacterial function under partial or fully anaerobic conditions, however, is challenging given the paucity of suitable live-cell imaging tools. Here, we introduce a series of RNA-based fluorescent biosensors that respond selectively to cyclic di-GMP, an intracellular bacterial second messenger that controls cellular motility and biofilm formation. We demonstrate the utility of these biosensors in vivo under both aerobic and anaerobic conditions, and we show that biosensor expression does not interfere with the native motility phenotype. Together, our results attest to the effectiveness and versatility of RNA-based fluorescent biosensors, priming further development and application of these and other analogous sensors to study host-microbial and microbial-microbial interactions through small molecule signals.
Asunto(s)
Técnicas Biosensibles/métodos , GMP Cíclico/análogos & derivados , ARN/metabolismo , Anaerobiosis , Aptámeros de Nucleótidos/metabolismo , GMP Cíclico/análisis , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Citometría de Flujo , Proteínas Fluorescentes Verdes/metabolismo , Movimiento , Fenotipo , Filogenia , Espectrometría de FluorescenciaRESUMEN
Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.
Asunto(s)
Fenómenos Electrofisiológicos , Geobacter/metabolismo , Nucleótidos Cíclicos/metabolismo , ARN Bacteriano/metabolismo , Riboswitch/fisiología , Sistemas de Mensajero Secundario/fisiología , Geobacter/genética , Nucleótidos Cíclicos/genética , ARN Bacteriano/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismoRESUMEN
Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive 'hypersensitive response' (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.
Asunto(s)
Resistencia a la Enfermedad/genética , Exones , Regulación de la Expresión Génica de las Plantas , Regiones Promotoras Genéticas , Transcripción Genética , Empalme Alternativo , Arabidopsis/genética , Proteínas Bacterianas/genética , Dexametasona/farmacología , Oomicetos/genética , Fenotipo , Plantas Modificadas Genéticamente/genética , Nicotiana/genética , Transcripción Genética/efectos de los fármacosRESUMEN
High-throughput enzyme activity screens are essential for target characterization and drug development, but few assays employ techniques or reagents that are applicable to both in vitro and live cell settings. Here, we present a class of selective and sensitive fluorescent biosensors for S-adenosyl-l-homocysteine (SAH) that provide a direct "mix and go" activity assay for methyltransferases (MTases), an enzyme class that includes several cancer therapeutic targets. Our riboswitch-based biosensors required an alternate inverted fusion design strategy, but retained full selectivity for SAH over its close structural analogue, the highly abundant methylation cofactor S-adenosyl-l-methionine (SAM). The level of ligand selectivity for these fluorescent biosensors exceeded that of commercial antibodies for SAH and proved critical to cellular applications, as we employed them to measure methylthioadenosine nucleosidase (MTAN) activity in live Escherichia coli. In particular, we were able to monitor in vivo increase of SAH levels upon chemical inhibition of MTAN using flow cytometry, which demonstrates high-throughput, single cell measurement of an enzyme activity associated with the biosynthesis of quorum sensing signal AI-2. Thus, this study presents RNA-based fluorescent biosensors as promising molecular reagents for high-throughput enzymatic assays that successfully bridge the gap between in vitro and in vivo applications.
Asunto(s)
Técnicas Biosensibles , Metiltransferasas/metabolismo , Riboswitch , S-Adenosilhomocisteína/análisis , Aptámeros de Nucleótidos , Escherichia coli/enzimología , Citometría de Flujo , Colorantes Fluorescentes , Ensayos Analíticos de Alto Rendimiento , Percepción de Quorum , Sensibilidad y EspecificidadRESUMEN
Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative (31)P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays.
Asunto(s)
Conformación de Ácido Nucleico , ARN/análisis , Riboswitch , Aptámeros de Nucleótidos/análisis , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Desnaturalización de Ácido Nucleico , ARN/química , ARN/metabolismo , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.
Asunto(s)
Técnicas Biosensibles , Fosfatos de Dinucleósidos/análisis , Fluorescencia , Listeria monocytogenes/citología , Listeria monocytogenes/metabolismo , ARN/química , Sistemas de Mensajero Secundario , Supervivencia Celular , Clostridioides difficile/enzimología , Fosfatos de Dinucleósidos/química , Activación Enzimática , Methanocaldococcus/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , RiboswitchRESUMEN
Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.