C-di-AMP, a bacterial second messenger, regulates various processes in Gram-positive bacteria and mycobacteria. Small molecule inhibitors of c-di-AMP metabolic enzymes could affect bacterial growth and viability. A medium throughput screening identified bromophenol-thiohydantoin (BTH) as the first inhibitor of c-di-AMP synthase, DisA. Herein, we performed SAR studies of bromophenol-thiohydantoin to identify the salient features on BTH that are important for DisA inhibition. Seemingly minor substitution changes (e.g., aromatic bromo to chloro substitutions) resulted in dramatic changes in ligand potency. Bromophenol TH is specific for c-di-AMP synthase and did not inhibit RocR (c-di-GMP PDE), YybT (c-di-AMP PDE) or WspR (c-di-GMP synthase).

Hydrated carbonyl groups in AI-2, a quorum sensing autoinducer, make key hydrogen bonding interactions in the binding site of LsrR (a transcriptional regulator). This can be recapitulated with geminal dibromides, via halogen bonding. Geminal dihalogens represent interesting isosteric replacements for hydrated carbonyls in ligands and are currently under-utilized in ligand design.

•Microfabricated heating device is combined with electrodes for microscale electrochemical measurements.•DNA melting curves were acquired via electrochemical means.•The microscale electrochemical platform facilitates the detection of single-nucleotide polymorphism.•Microfabrication method enables microwell array fabrication.Microfabrication methods have been used to fabricate a new microscale platform that integrates thermal control and multi-electrode components to enable rapid, temperature-dependent electrochemical measurements on small-volume fluid samples. A wide range of biochemical phenomena can be characterized with the device, for example, when monitoring interactions at the working electrode between probe and target species which include an electroactive moiety. Employing square wave voltammetry, we have demonstrated the utility and reproducibility of the microplatform in melting studies on full-match, single-mismatch, and double-mismatch DNA structures of relevance to single-nucleotide polymorphism (SNP) discrimination. As shown, the small size of the reported device, low volume for the samples it can interrogate (∼10 μL), individual addressing of platform components and fast localized heating (settling times ∼5 s) combine to allow for efficient sample analyses. In addition, a straight-forward route exists, involving replication into array formats and integration with microfluidics, for extending the technology toward eventual high throughput work on drug discovery and medical diagnostics.

Synthetic molecules that modulate quorum sensing, QS, in bacteria have great potential for use in synthetic biology applications as well as acting as anti-virulence and anti-biofilm agents. Acylhomoserine lactone (AHL)-based autoinducer analogs have been extensively developed as QS modulators but these suffer from both chemical and enzymatic degradations. Here, we reveal that 3-aminooxazolidinone acylhomoserine lactone analogs are hydrolytically stable and are as potent in activating LuxR-type receptors. Docking analysis revealed that 3-oxo-C12-3-aminooxazolidinone docked in LasR of P. aeruginosa, making similar interactions with the protein's active-site residues to the native ligand, 3-oxo-C12 HSL. Experimentally, 3-oxo-C12-3-aminooxazolidinone was equally as potent as the natural ligand in inducing bioluminescence in E. coli carrying a bioluminescent gene that was under the control of LasR. In C. violaceum CV026, the 3-aminooxazolidinone analogs could also modulate pigment (violacein) formation, albeit this time not as potent as the natural AHL ligands.

c-di-AMP is an important bacterial second messenger found in Gram-positive and mycobacteria. c-di-AMP regulates myriads of processes in bacteria as well as immune response in higher organisms so interest in small molecules that would attenuate the activity of c-di-AMP metabolism enzymes is high. Herein, we report the first small molecule inhibitor of a c-di-AMP synthase, DisA, using a coralyne-based assay.

The article by Gerdt and colleagues in this issue of Chemistry & Biology provides molecular insights into how nonlactone quorum sensing modulators either activate or deactivate LasR. Interestingly, an antagonist could flip into an agaonist upon mutation of a single residue in the autoinducer binding site.

Cyclic diadenosine monophosphate (c-di-AMP) has emerged as an important dinucleotide that is involved in several processes in bacteria, including cell wall remodeling (and therefore resistance to antibiotics that target bacterial cell wall). Small molecules that target c-di-AMP metabolism enzymes have the potential to be used as antibiotics. Coralyne is known to form strong complexes with polyadenine containing eight or more adenine stretches but not with short polyadenine oligonucleotides. Using a panel of techniques (UV, both steady state fluorescence and fluorescence lifetime measurements, circular dichroism (CD), NMR, and Job plots), we demonstrate that c-di-AMP, which contains only two adenine bases is an exception to this rule and that it can form complexes with coralyne, even at low micromolar concentrations. Interestingly, pApA (the linear analog of c-di-AMP that also contains two adenines) or cyclic diguanylate (c-di-GMP, another nucleotide second messenger in bacteria) did not form any complex with coralyne. Unlike polyadenine, which forms a 2:1 complex with coralyne, c-di-AMP forms a higher order complex with coralyne (≥6:1). Additionally, whereas polyadenine reduces the fluorescence of coralyne when bound, c-di-AMP enhances the fluorescence of coralyne. We use the quenching property of halides to selectively quench the fluorescence of unbound coralyne but not that of coralyne bound to c-di-AMP. Using this simple selective quenching strategy, the assay could be used to monitor the synthesis of c-di-AMP by DisA or the degradation of c-di-AMP by YybT. Apart from the practical utility of this assay for c-di-AMP research, this work also demonstrates that, when administered to cells, intercalators might not only associate with polynucleotides, such as DNA or RNA, but also could associate with cyclic dinucleotides to disrupt or modulate signal transduction processes mediated by these nucleotides.

G-quadruplexes have shown great promise as chemotherapeutic targets, probably by inhibiting telomere elongation or downregulating oncogene expression. There have been many G-quadruplex ligands developed over the years but only a few have drug-like properties. Consequently only a few G-quadruplex ligands have entered clinical trials as cancer chemotherapeutic agents. The DNA minor groove ligand, berenil (diminazene aceturate or DMZ), is used to treat animal trypanosomiasis and hence its toxicological profile is already known, making it an ideal platform to engineer into new therapeutics. Herein, using a plethora of biophysical methods including UV, NMR, MS and ITC, we show that DMZ binds to several G-quadruplexes with a Kd of ∼1 nM. This is one of the strongest G-quadruplex binding affinities reported to date and is 103 tighter than the berenil affinity for an AT-rich duplex DNA. Structure–activity-relationship studies demonstrate that the two amidine groups on DMZ are important for binding to both G-quadruplex and duplex DNA. This work reveals that DMZ or berenil is not as selective for AT-rich duplexes as originally thought and that some of its biological effects could be manifested through G-quadruplex binding. The DMZ scaffold represents a good starting point to develop new G-quadruplex ligands for cancer cell targeting.

Cyclic dinucleotides have emerged as second messengers that regulate diverse processes in bacteria, as well as regulating the production of type I interferons in metazoans. Fluorescent sensors for these important second messengers are highly sought-after for high-throughput inhibitor discovery, yet most sensors reported to date are not amenable for high-throughput screening purposes. Herein, we demonstrate that a new analog, 3′,3′-cG(d2AP)MP, which is a 2-aminopurine (2AP)-containing cyclic dinucleotide, self-associates in the presence of Mn2+ with an association constant of 120000 M−1. 3′3′-cG(d2AP)MP can also form a heterodimer with cGAMP, activator of immune regulator, STING, or the bacterial biofilm regulator, c-di-GMP in the presence of Mn(II). Upon dimer formation, the fluorescence of 3′,3′-cG(d2AP)MP is quenched and this provides a convenient method to monitor the enzymatic processing of both DGC and PDE enzymes, opening up several opportunities for the discovery of inhibitors of nucleotide signaling.

This review highlights various methods that can be used for a sensitive detection of nucleic acids without using thermal cycling procedures, as is done in PCR or LCR. Topics included are nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated amplification (LAMP), Invader assay, rolling circle amplification (RCA), signal mediated amplification of RNA technology (SMART), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA), nicking endonuclease signal amplification (NESA) and nicking endonuclease assisted nanoparticle activation (NENNA), exonuclease-aided target recycling, Junction or Y-probes, split DNAZyme and deoxyribozyme amplification strategies, template-directed chemical reactions that lead to amplified signals, non-covalent DNA catalytic reactions, hybridization chain reactions (HCR) and detection via the self-assembly of DNA probes to give supramolecular structures. The majority of these isothermal amplification methods can detect DNA or RNA in complex biological matrices and have great potential for use at point-of-care.

C-di-GMP has emerged as a ubiquitous second messenger, which regulates the transition between sessile and motile lifestyles and virulence factor expression in many pathogenic bacteria using both RNA riboswitches and protein effectors. We recently showed that two additional class I c-di-GMP riboswitch aptamers (Ct-E88 and Cb-17B) bind c-di-GMP with nanomolar affinity, and that Ct-E88 RNA binds 2′-F-c-di-GMP 422 times less tightly than class I Vc2 RNA. Based on sequence comparison, it was concluded that the global folds of Ct-E88 and Vc2 RNAs were similar and that differences in ligand binding were probably due to differences in binding site architectures. Herein, we utilized EMSA, aptamer sensing spinach modules, SAXS and 1D NMR titration to study the conformational transitions of Ct-E88. We conclude that whereas the global folds of the bound states of Vc2 and Ct-E88 RNAs are similar, the unbound states are different and this could explain differences in ligand affinities between these class I c-di-GMP riboswitches.

For an organism to survive, it must be able to sense its environment and regulate physiological processes accordingly. Understanding how bacteria integrate signals from various environmental factors and quorum sensing autoinducers to regulate the metabolism of various nucleotide second messengers c-di-GMP, c-di-AMP, cGMP, cAMP and ppGpp, which control several key processes required for adaptation is key for efforts to develop agents to curb bacterial infections. In this review, we provide an update of nucleotide signaling in bacteria and show how these signals intersect or integrate to regulate the bacterial phenotype. The intracellular concentrations of nucleotide second messengers in bacteria are regulated by synthases and phosphodiesterases and a significant number of these metabolism enzymes had been biochemically characterized but it is only in the last few years that the effector proteins and RNA riboswitches, which regulate bacterial physiology upon binding to nucleotides, have been identified and characterized by biochemical and structural methods. C-di-GMP, in particular, has attracted immense interest because it is found in many bacteria and regulate both biofilm formation and virulence factors production. In this review, we discuss how the activities of various c-di-GMP effector proteins and riboswitches are modulated upon c-di-GMP binding. Using V. cholerae, E. coli and B. subtilis as models, we discuss how both environmental factors and quorum sensing autoinducers regulate the metabolism and/or processing of nucleotide second messengers. The chemical syntheses of the various nucleotide second messengers and the use of analogs thereof as antibiofilm or immune modulators are also discussed.

C-di-GMP is a second messenger in bacteria and partly regulates bacterial physiology by binding to class I and II riboswitches. Four class I c-di-GMP riboswitch aptamer candidates, Ct-E88, Cb-17B, Cb-E43 and Cd-630 RNAs, selected from a GEMM RNA sequence motif in the Rfam database, were expressed and experimentally verified to bind to c-di-GMP. The two newly characterized c-di-GMP riboswitches, Ct-E88 and Cb-E43, bound c-di-GMP with nanomolar Kd whereas the affinities of Cb-17B and Cd-630 for c-di-GMP were at least a 100-fold weaker. Interestingly, whereas the three riboswitches (Vc2, Et-E88 and Cb-E43) bound c-di-GMP with similar Kd values, 2′-modified analogs of c-di-GMP differentially bound to these three class I aptamers. For example, 2′-F-c-di-GMP bound Vc2 with a Kd value of 102 nM whereas the Kd value of 2′-F-c-di-GMP–Ct-E88 is 43 μM (422× higher than that for Vc2 RNA), revealing that there are differences in the binding sites of functional class I c-di-GMP riboswitches.

Junction probe (JP) platform is an isothermal endonuclease-based detection assay for both RNA and DNA. Herein, we screen 31 REAse and identify effective restriction endonucleases that can be used for JP detection. Secondly, we investigate how different probe architectures affect JP cleavage rates and conclude that although molecular beacon (MB) JP probes give less background noise than linear JP probes, the cleavage of MB JP probes are slower than linear JP probes.

Cyclic-di-GMP (c-di-GMP) is a central regulator of bacterial behavior. Various studies have implicated c-di-GMP in biofilm formation and virulence factor production in multitudes of bacteria. Hence it is expected that the disruption of c-di-GMP signaling could provide an effective means to disrupt biofilm and/or virulence factor formation in several bacteria of clinical relevance. C-di-GMP achieves the regulation of bacterial phenotype via binding to several effector molecules including transcription factors, enzymes and riboswitches. Crystal structure analyses of c-di-GMP effector molecules, in complex with the ligand, reveal that various classes of c-di-GMP receptors recognize this dinucleotide using different sets of recognition elements. Therefore, it is plausible that different analogues of c-di-GMP could be used to selectively modulate a specific class of c-di-GMP binding receptors, and hence modulate the bacterial phenotype. Thus far only a detailed study of the differential binding of c-di-GMP analogues to riboswitches, but not proteins, has been reported. In this report, we prepared various 2′-modified analogues of c-di-GMP and studied both polymorphisms of these analogues using DOSY NMR and the binding to several effector proteins, such as PilZ-containing proteins, diguanylate cyclases (DGC) containing I-sites, and phoshphodiesterases (PDE). 2′-Modification of c-di-GMP did not adversely affect the propensity to form higher aggregates, such as octameric forms, in the presence of potassium salts. Interestingly, we find that the selective binding to different classes of c-di-GMP binding proteins could be achieved with the 2′-modified analogues and that 2′-F analogue of c-di-GMP binds to the I-site of DGCs better (four times) than the native dinucleotide, c-di-GMP, whereas c-di-GMP binds to PDEs better (10 times) than 2′-F-c-di-GMP. 2′-F-c-di-GMP potently inhibits c-di-GMP synthesis by DGCs and hence raises the potential that cell permeable analogues of 2′-F-c-di-GMP could be used to disrupt c-di-GMP signaling in bacteria.

We report a facile approach to a cyclopropyl-fused pyrrolidine, which contains four stereogenic centers, by employing the N–O tethered carbenoid methodology. The synthesis was facilitated by the development of a direct Mitsunobu reaction of alcohols with N-alkyl-N-hydroxyl amides to give diazo precursors, which upon intramolecular cyclopropanation yielded a library of N–O containing cyclopropyl-fused bicyclic intermediates. Elaboration of the N–O moiety of one member of this library resulted in the formation of the desired pyrrolidine ring demonstrating the potential of this methodology for making cyclopropyl-fused heterocycles.

A methylene-blue (MB)-labeled molecular beacon junction probe allows for a signal-on electrochemical detection of nucleic acids via target recycling using endonucleases. Electron transfer is reduced when the MB is intercalated in the stem of the molecular beacon, but then electron transfer from MB to a gold electrode is enhanced upon cleavage of the junction probe due to increased probability of MB approaching the electrode when attached to the more flexible ssDNA.

C-di-GMP regulates important processes involved in biofilm formation and virulence factors production in several bacteria. Herein we report a simple fluorescent strategy that allows for the detection of c-di-GMP (as low as 320 nM) using a Vc2 class I riboswitch domain as the sensing region and spinach as the fluorescent reporting module.

Cations affect the topology and enzymatic proficiency of most macromolecular catalysts but the role of cations in DNAzyme peroxidation reactions remains unresolved. Herein, we use statistical methods (ANOVA, t-test and Wilcoxon Mann–Whitney non-parametric test) to demonstrate that there are strong associations between cations, DNAzyme topology, peroxidation substrate and peroxidation rates of G-quadruplex peroxidises. Ammonium cation was found to be superior to all tested cations, including potassium. A t-test indicated that NH4+ was better than K+ with a p-value = 0.05. Interestingly, the nature of the peroxidation substrate employed affected the dependence of peroxidation rate on the cation present and of the three substrates tested, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), tyramine and 3,3′,5,5′-tetramethylbenzidine (TMB), ABTS was the most sensitive to the nature of cation present.Graphical abstractHighlights► G-quadruplex peroxidation rate depends on the cation present. ► NH4+ is best cation amongst Li+, Na+, K+, Cs+, Rb+, Sr2+, Br2+, Pb2+ and Tb3+. ► ANOVA reveals cation effect is dependent on DNAzyme topology and peroxidation substrate.

There have been intensive efforts to find small molecule antagonists for bacterial quorum sensing (QS) mediated by the “universal” QS autoinducer, AI-2. Previous work has shown that linear and branched acyl analogues of AI-2 can selectively modulate AI-2 signaling in bacteria. Additionally, LsrK-dependent phosphorylated analogues have been implicated as the active inhibitory form against AI-2 signaling. We used these observations to synthesize an expanded and diverse array of AI-2 analogues, which included aromatic as well as cyclic C-1-alkyl analogues. Species-specific analogues that disrupted AI-2 signaling in Escherichia coli and Salmonella typhimurium were identified. Similarly, analogues that disrupted QS behaviors in Pseudomonas aeruginosa were found. Moreover, we observed a strong correlation between LsrK-dependent phosphorylation of these acyl analogues and their ability to suppress QS. Significantly, we demonstrate that these analogues can selectively antagonize QS in single bacterial strains in a physiologically relevant polymicrobial culture.

C-di-GMP has emerged as a signalling molecule that regulates a variety of processes in several bacteria; therefore there is interest in the development of biotinylated analogs for the identification of binding partners. No detailed study has been done to evaluate if biotinylated analogs of c-di-GMP are capable of binding to c-di-GMP receptors. Herein, we evaluate the binding of commercially available 2′-biotinylated c-di-GMP and phosphorothioate 2′-biotinylated c-di-GMP, prepared via a facile solid-phase synthesis, to several c-di-GMP receptors. Docking, using Autodock vina software, as well as experimental studies of these analogs, with c-di-GMP class I and II riboswitches and binding proteins, reveal that some, but not all, c-di-GMP receptors can tolerate the 2′-modification of c-di-GMP with biotin.

Herein, we demonstrate that the bacterial signaling molecule, c-di-GMP, can enhance the peroxidation of hemin when proflavine is present. The c-di-GMP–proflavine–hemin nucleotidezyme can oxidize the colorless compound 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, to the colored radical cation ABTS˙+ and hence provides simple colorimetric detection of c-di-GMP at low micromolar concentrations.

C-di-GMP has emerged as an important bacterial signaling molecule that is involved in biofilm formation. Small molecules that can form biologically inactive complexes with c-di-GMP have the potential to be used as anti-biofilm agents. Herein, we report that water-soluble diamidinium/iminium aromatics (such as berenil), which are traditionally considered as minor groove binders of nucleic acids, are capable of aggregating c-di-GMP into G-quadruplexes via π-stacking interactions.

Recently, there has been an explosion of research activities in the cyclic dinucleotides field. Cyclic dinucleotides, such as c-di-GMP and c-di-AMP, have been shown to regulate bacterial virulence and biofilm formation. c-di-GMP can exist in different aggregate forms, and it has been demonstrated that the polymorphism of c-di-GMP is influenced by the nature of cation that is present in solution. In previous work, polymorphism of c-di-GMP could only be demonstrated at hundreds of micromolar concentrations of the dinucleotide, and it has been a matter of debate if polymorphism of c-di-GMP exists under in vivo conditions. In this Article, we demonstrate that c-di-GMP can form G-quadruplexes at low micromolar concentrations when aromatic molecules such as thiazole orange template the quadruplex formation. We then use this property of aromatic molecule-induced G-quadruplex formation of c-di-GMP to design a thiazole orange-based fluorescent detection of this important signaling molecule. We determine, using this thiazole orange assay on a crude bacterial cell lysate, that WspR D70E (a constitutively activated diguanylate cyclase) is functional in vivo when overexpressed in E. Coli. The intracellular concentration of c-di-GMP in an E. Coli cell that is overexpressed with WspR D70E is very high and can reach 2.92 mM.

The cyclic dinucleotide c-di-GMP is a master regulator of bacterial virulence and biofilm formation. The activations of c-di-GMP metabolism proteins, diguanylate cyclases (DGCs) and phosophodiesterases (PDEs), usually lead to diametrically opposite phenotypes in bacteria. Analogues of c-di-GMP, which can selectively modulate the activities of c-di-GMP processing proteins, will be useful chemical tools for studying and altering bacterial behavior. Herein we report that a conservative modification of one of the phosphate groups in c-di-GMP with a bridging sulfur in the phosphodiester linkage affords an analogue called endo-S-c-di-GMP. Computational, NMR (including DOSY), and CD experiments all reveal that, unlike c-di-GMP, endo-S-c-di-GMP does not readily form higher aggregates. The lower propensity of endo-S-c-di-GMP to form aggregates (as compared to that of c-di-GMP) is probably due to a higher activation barrier to convert from the “open” conformer (where the two guanines are on opposite faces) to the “closed” conformer (where the two guanines are on the same face). Consequently, endo-S-c-di-GMP has selectivity for proteins that bind monomeric but not dimeric c-di-GMP, which form from the “closed” conformer. For example, endo-S-c-di-GMP can inhibit the hydrolysis of c-di-GMP by RocR (a PDE enzyme that binds monomeric c-di-GMP) but did not bind to Alg44 (a PilZ protein) or regulate WspR (a DGC enzyme that has been shown to bind to dimeric c-di-GMP). This work demonstrates that selective binding to different classes of c-di-GMP binding proteins could be achieved by altering analogue conformer populations (conformational steering). We provide important design principles for the preparation of selective PDE inhibitors and reveal the role played by the c-di-GMP backbone in c-di-GMP polymorphism and binding to processing proteins.

The ubiquitous bacterial biofilm regulator, c-di-GMP can form G-quadruplexes at physiological conditions in the presence of some aromatic compounds, such as acriflavine and proflavine. The fluorescence of these compounds is quenched upon c-di-GMP binding and some of the formed c-di-GMP G-quadruplexes are stable even at 75 °C.

Herein, we demonstrate how to detect nucleic acids that do not contain restriction endonuclease recognition sites with restriction endonucleases. We show that the topology of DNA probes used in this detection strategy remarkably affects the efficiency of RNA/DNA detection.

The widespread use of antibiotics and the emergence of resistant strains call for new approaches to treat bacterial infection. Bacterial cell−cell communication or “quorum sensing” (QS) is mediated by “signatures” of small molecules that represent targets for “quenching” communication and avoiding virulent phenotypes. Only a handful of small molecules that antagonize the action of the “universal” autoinducer, AI-2, have been reported. The biological basis of antagonism, as well as the targets for these select few AI-2 antagonists, have not been clearly defined. We have developed C-1 alkyl analogs of AI-2 that quench the QS response in multiple bacterial species simultaneously. We also demonstrate the biological basis for this action. Like AI-2, the analogs are activated by the bacterial kinase, LsrK, and modulate AI-2 specific gene transcription through the transcriptional regulator, LsrR. Interestingly, addition of a single carbon to the C1-alkyl chain of the analog plays a crucial role in determining the effect of the analog on the QS response. While an ethyl modified analog is an agonist, propyl becomes an antagonist of the QS circuit. In a trispecies synthetic ecosystem comprised of E. coli, S. typhimurium, and V. harveyi we discovered both cross-species and species-specific anti-AI-2 QS activities. Our results suggest entirely new modalities for interrupting or tailoring the network of communication among bacteria.

The identification of a suitable non-fluorescent molecule that can be oxidized by G-quadruplexes into a fluorescent product will be important for several fields such as bioanalyte sensing and cancer therapeutic discovery. Herein, we demonstrate that 2′,7′-dichlorodihydrofluorescein diacetate is a superior reducing substrate for the fluorometric detection of bioanalytes using peroxidase-mimicking G-quadruplexDNAzymes.

Split G-rich DNA probes can assemble into active peroxidase-mimicking DNAzymes in the presence of bioanalytes such as DNA, thereby providing a simple and cheap means to detect analytes in biological samples. A comprehensive study designed to reveal the salient probe architectural features and reaction conditions that facilitate facile reconstitution into enzymatically proficient enzymes unveiled these important findings: (a) The loops that connect the G3-tracts in a G-quadruplex structure can be replaced with a stem-loop or loop-stem-loop motif without destabilizing the resulting quadruplex structure; endowing the split G-rich probes with regions of limited complementarity leads to more proficient reconstituted enzymes. (b) The addition of hemin to antiparallel G-quadruplex DNAzymes lead to a blue shift in the CD spectra of the G-quadruplex DNAzymes. (c) The architectures of the DNA motifs that lie adjacent to the G-quadruplex structure influence both the stability and the enzymatic proficiency of the reconstituted enzymes. (d) The nature of the monovalent cation that is present in excess is a key determinant of the turnovernumber of the G-quadruplex DNAzyme; decomposition of G-quadruplex DNAzymes is slower in buffers that contain ammonium ions than those that contain sodium or potassium ions. These findings are important for the design of bioassays that use peroxidase-mimicking G-quadruplexes as detection labels.

C1-alkyl AI-2 analogues do not induce bioluminescence in V. harveyi on their own but enhance the bioluminescence induced by AI-2 in a synergistic fashion. A new facile synthesis of AI-2 facilitates the synthesis of a diverse set of AI-2 analogues and biological screening suggests that receptors that are involved in the synergistic bioluminescence production in V. harveyi are promiscuous.

Cyclic-di-guanylate (c-di-GMP) has emerged as a general and important signaling molecule uniquely present in bacteria: herein we provide a simple solid-phase synthesis of c-di-GMP using an automated DNA synthesizer for the majority of the synthesis.

In the last decade, there has been an explosion in the use of G-quadruplex labels to detect various analytes, including DNA/RNA, proteins, metals and other metabolites. In this review, we focus on strategies for the detection of nucleic acids, using G-quadruplexes as detection labels or as enzyme labels that amplify detection signals. Methods to detect other analytes are briefly mentioned. We highlight various strategies, including split G-quadruplex, hemin–G-quadruplex conjugates, molecular beacon G-quadruplex or inhibited G-quadruplex probes. The tandem use of G-quadruplex labels with various DNA-modifying enzymes, such as polymerases (used for rolling circle amplification), exonucleases and endonucleases, is also discussed. Some of the detection modalities that are discussed in this review include fluorescence, colorimetric, chemiluminescence, and electrochemical methods.

Herein we show that two isothermal amplification strategies, rolling circle amplification and junction probe strategy, can be used in tandem in the same tube under isothermal conditions to detect HPV16 in clinical cervical swabs. It was discovered that the prior treatment of the clinical sample with a cocktail of restriction endonucleases (REAses) to digest the genomic DNA facilitated the isothermal detection assay.

C-di-GMP regulates important processes involved in biofilm formation and virulence factors production in several bacteria. Herein we report a simple fluorescent strategy that allows for the detection of c-di-GMP (as low as 320 nM) using a Vc2 class I riboswitch domain as the sensing region and spinach as the fluorescent reporting module.

Hydrated carbonyl groups in AI-2, a quorum sensing autoinducer, make key hydrogen bonding interactions in the binding site of LsrR (a transcriptional regulator). This can be recapitulated with geminal dibromides, via halogen bonding. Geminal dihalogens represent interesting isosteric replacements for hydrated carbonyls in ligands and are currently under-utilized in ligand design.

Herein, we demonstrate how to detect nucleic acids that do not contain restriction endonuclease recognition sites with restriction endonucleases. We show that the topology of DNA probes used in this detection strategy remarkably affects the efficiency of RNA/DNA detection.

For an organism to survive, it must be able to sense its environment and regulate physiological processes accordingly. Understanding how bacteria integrate signals from various environmental factors and quorum sensing autoinducers to regulate the metabolism of various nucleotide second messengers c-di-GMP, c-di-AMP, cGMP, cAMP and ppGpp, which control several key processes required for adaptation is key for efforts to develop agents to curb bacterial infections. In this review, we provide an update of nucleotide signaling in bacteria and show how these signals intersect or integrate to regulate the bacterial phenotype. The intracellular concentrations of nucleotide second messengers in bacteria are regulated by synthases and phosphodiesterases and a significant number of these metabolism enzymes had been biochemically characterized but it is only in the last few years that the effector proteins and RNA riboswitches, which regulate bacterial physiology upon binding to nucleotides, have been identified and characterized by biochemical and structural methods. C-di-GMP, in particular, has attracted immense interest because it is found in many bacteria and regulate both biofilm formation and virulence factors production. In this review, we discuss how the activities of various c-di-GMP effector proteins and riboswitches are modulated upon c-di-GMP binding. Using V. cholerae, E. coli and B. subtilis as models, we discuss how both environmental factors and quorum sensing autoinducers regulate the metabolism and/or processing of nucleotide second messengers. The chemical syntheses of the various nucleotide second messengers and the use of analogs thereof as antibiofilm or immune modulators are also discussed.

C1-alkyl AI-2 analogues do not induce bioluminescence in V. harveyi on their own but enhance the bioluminescence induced by AI-2 in a synergistic fashion. A new facile synthesis of AI-2 facilitates the synthesis of a diverse set of AI-2 analogues and biological screening suggests that receptors that are involved in the synergistic bioluminescence production in V. harveyi are promiscuous.

The ubiquitous bacterial biofilm regulator, c-di-GMP can form G-quadruplexes at physiological conditions in the presence of some aromatic compounds, such as acriflavine and proflavine. The fluorescence of these compounds is quenched upon c-di-GMP binding and some of the formed c-di-GMP G-quadruplexes are stable even at 75 °C.

A methylene-blue (MB)-labeled molecular beacon junction probe allows for a signal-on electrochemical detection of nucleic acids via target recycling using endonucleases. Electron transfer is reduced when the MB is intercalated in the stem of the molecular beacon, but then electron transfer from MB to a gold electrode is enhanced upon cleavage of the junction probe due to increased probability of MB approaching the electrode when attached to the more flexible ssDNA.

c-di-AMP is an important bacterial second messenger found in Gram-positive and mycobacteria. c-di-AMP regulates myriads of processes in bacteria as well as immune response in higher organisms so interest in small molecules that would attenuate the activity of c-di-AMP metabolism enzymes is high. Herein, we report the first small molecule inhibitor of a c-di-AMP synthase, DisA, using a coralyne-based assay.