Herman O. Sintim

Colorimetric Split G-Quadruplex Probes for Nucleic Acid Sensing: Improving Reconstituted DNAzyme's Catalytic Efficiency via Probe Remodeling

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 turnover number 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.

Paradigm shift in discovering next-generation anti-infective agents: targeting quorum sensing, c-di-GMP signaling and biofilm formation in bacteria with small molecules

Small molecules that can attenuate bacterial toxin production or biofilm formation have the potential to solve the bacteria resistance problem. Although several molecules, which inhibit bacterial cell-to-cell communication (quorum sensing), biofilm formation and toxin production, have been discovered, there is a paucity of US FDA-approved drugs that target these processes. Here, we review the current understanding of quorum sensing in important pathogens such as Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus and provide examples of experimental molecules that can inhibit both known and unknown targets in bacterial virulence factor production and biofilm formation. Structural data for protein targets that are involved in both quorum sensing and cyclic diguanylic acid signaling are needed to aid the development of molecules with drug-like properties in order to target bacterial virulence factors production and biofilm formation.

Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions

Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interactions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interactions without the need to purify the protein when performed in cells with low background activity. This method, differential radial capillary action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein–ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through capillary action. We show here that DRaCALA allows detection of specific interactions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli, demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interaction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interactions in a systematic and comprehensive approach.

Junction Probes : Sequence Specific Detection of Nucleic Acids via Template Enhanced Hybridization Processes

Junction probe nucleic acid detection technology allows the amplified sensing of analytes at isothermal conditions. The addition of a second dimension to detection probes permits the use of cheap commercially available DNA processing enzymes such as restriction endonucleases to detect single nucleotide polymorphisms, SNPs.

The difluorotoluene debate--a decade later

2,4-Difluorotoluene is unusual among hydrofluorocarbons because it is shaped like the DNA base thymine. It was first synthesised as a nucleotide analogue and incorporated into DNA a decade ago. Although it is a nonpolar molecule, it was found to be replicated by DNA polymerase enzymes as if it were thymine. We concluded that replication of DNA base pairs can occur without Watson-Crick hydrogen bonds, and hypothesised that steric effects, rather than these hydrogen bonds, were the main arbiters of DNA replication fidelity. A debate was initiated then, with claims by some that the molecule is polar and forms hydrogen bonds with adenine, thus supporting the hydrogen bonding theory of DNA replication. Here we discuss the evolution of this debate, and reflect on the relevant data that have since come from hundreds of papers and dozens of laboratories. Although discussion on this topic continues, the steric hypothesis for DNA replication is now widely accepted among biochemists, and the changing paradigm has been reflected in textbooks.

STING Ligand c-di-GMP Improves Cancer Vaccination against Metastatic Breast Cancer

Chandra, Quispe-Tintaya, and colleagues show that stimulator of IFN genes (STING) ligand c-di-GMP activated caspase-3, stimulated T cells, and nearly completely eliminated all metastases in mouse breast cancer model 4T1, when combined with Listeria monocytogenes–based Mage-b vaccine in a therapeutic setting. Cancer vaccination may be our best and most benign option for preventing or treating metastatic cancer. However, breakthroughs are hampered by immune suppression in the tumor microenvironment. In this study, we analyzed whether cyclic diguanylate (c-di-GMP), a ligand for stimulator of interferon genes (STING), could overcome immune suppression and improve vaccination against metastatic breast cancer. Mice with metastatic breast cancer (4T1 model) were therapeutically immunized with an attenuated Listeria monocytogenes (LM)–based vaccine, expressing tumor-associated antigen Mage-b (LM-Mb), followed by multiple low doses of c-di-GMP (0.2 μmol/L). This treatment resulted in a striking and near elimination of all metastases. Experiments revealed that c-di-GMP targets myeloid-derived suppressor cells (MDSC) and tumor cells. Low doses of c-di-GMP significantly increased the production of IL12 by MDSCs, in correlation with improved T-cell responses to Mage-b, whereas a high dose of c-di-GMP (range, 0.3–3 mmol/L) activated caspase-3 in the 4T1 tumor cells and killed the tumor cells directly. On the basis of these results, we tested one administration of high-dose c-di-GMP (3 mmol/L) followed by repeated administrations of low-dose c-di-GMP (0.2 μmol/L) in the 4T1 model, and found equal efficacy compared with the combination of LM-Mb and c-di-GMP. This finding correlated with a mechanism of improved CD8 T-cell responses to tumor-associated antigens (TAA) Mage-b and Survivin, most likely through cross-presentation of these TAAs from c-di-GMP–killed 4T1 tumor cells, and through c-di-GMP–activated TAA-specific T cells. Our results demonstrate that activation of STING-dependent pathways by c-di-GMP is highly attractive for cancer immunotherapy. Cancer Immunol Res; 2(9); 901–10. ©2014 AACR.

Agents that inhibit bacterial biofilm formation

In the biofilm form, bacteria are more resistant to various antimicrobial treatments. Bacteria in a biofilm can also survive harsh conditions and withstand the hosts immune system. Therefore, there is a need for new treatment options to treat biofilm-associated infections. Currently, research is focused on the development of antibiofilm agents that are nontoxic, as it is believed that such molecules will not lead to future drug resistance. In this review, we discuss recent discoveries of antibiofilm agents and different approaches to inhibit/disperse biofilms. These new antibiofilm agents, which contain moieties such as imidazole, phenols, indole, triazole, sulfide, furanone, bromopyrrole, peptides, etc. have the potential to disperse bacterial biofilms in vivo and could positively impact human medicine in the future.

Nanomolar fluorescent detection of c-di-GMP using a modular aptamer strategy

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.

Thiazole Orange-Induced c-di-GMP Quadruplex Formation Facilitates a Simple Fluorescent Detection of This Ubiquitous Biofilm Regulating Molecule

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.

Evidence of magnetic isotope effects during thermochemical sulfate reduction

Thermochemical sulfate reduction experiments with simple amino acid and dilute concentrations of sulfate reveal significant degrees of mass-independent sulfur isotope fractionation. Enrichments of up to 13‰ for 33S are attributed to a magnetic isotope effect (MIE) associated with the formation of thiol-disulfide, ion-radical pairs. Observed 36S depletions in products are explained here by classical (mass-dependent) isotope effects and mixing processes. The experimental data contrasts strongly with multiple sulfur isotope trends in Archean samples, which exhibit significant 36S anomalies. These results support an origin other than thermochemical sulfate reduction for the mass-independent signals observed for early Earth samples.