Systematic Investigations of Different Cytosine Modifications on CpG Dinucleotide Sequences: The Effects on the B‑Z Transition

We have first demonstrated the distinctive effects of three newly reported epigenetic modifications, including 5hmC, 5fC, and 5caC, on B-Z transition of CpG dinucleotide DNAs. We have performed detailed assays and compared their effects. We further studied the regulation of B-Z transition of CpG dinucleotide dodecamers by alternating oxidation and alternating reduction

Systematic Investigations of Different Cytosine Modifications on CpG Dinucleotide Sequences: The Effects on the B‑Z Transition

We have first demonstrated the distinctive effects of three newly reported epigenetic modifications, including 5hmC, 5fC, and 5caC, on B-Z transition of CpG dinucleotide DNAs. We have performed detailed assays and compared their effects. We further studied the regulation of B-Z transition of CpG dinucleotide dodecamers by alternating oxidation and alternating reduction

Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection

G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H₂O₂ generation from O₂ and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications

Serratamolide purification and verification of biological activity. A.

<p>Structure of serratamolide. <b>B.</b> HPLC trace of spent supernatants from a <i>swrW</i> mutant with either an empty vector (<i>swrW</i>+vector) or a <i>swrW</i> expression plasmid (<i>swrW</i>+p<i>swrW</i>). The expected peak for serratamolide is indicated by an arrow. <b>C.</b> Swarming motility of an <i>swrW</i> mutant treated with DMSO or purified serratamolide. This shows that the purified compound restores swarming motility as expected.</p

Isolation of <i>swrW</i> and its role in surfactant production and hemolysis. A.

<p>Sample genetic screen plate shows <i>crp</i> mutants with random transposon insertions. The white arrow indicates a colony deficient in secreted hemolysis production with a transposon insertion that mapped to the <i>swrW</i> gene. This image is illuminated from the back, so that the gold surface coloration is not apparent. <b>B.</b> Surface coloration of <i>crp swrW</i> double mutants is metallic gold compared to the red-orange color of the <i>crp</i> mutant. <b>C.</b> Surfactant zones (mm) measured from the colony to the maximum extent of the surfactant zone (n≥4 per genotype). Asterisk represents a statistically significant increase in surfactant zone compared to the WT (p<0.05) by ANOVA with Tukey’s post-test. <b>D.</b> Mutation of <i>swrW</i> reduced or eliminated the ability of laboratory strain Nima and three of five clinical keratitis isolates to make zones of hemolysis on blood agar plates. Representative images from reproducible experiments are shown.</p

Genetic evidence that serratamolide mediates hemolysis. A.

<p>Hemolysis and swarming by a mutant known to have elevated serratamolide production (<i>hexS</i>) is increased, and these phenotypes require SwrW. <b>B.</b> Elevated expression of a <i>swrW</i> promoter reporter in the <i>crp</i> mutant. Top, expression measured using a plasmid based-<i>tdtomato</i> reporter construct at t = 20 hrs. Asterisk indicates statistical significance (p<0.05) by the Student’s T-test. A representative experiment is shown (n = 4). Error bars indicate one standard deviation. Bottom, semi-quantitative RT-PCR analysis of RNA from WT and Δ<i>crp</i> mutant strains measured relative expression of <i>swrW</i> and internal standard 16S RNA from stationary phase cultures (OD₆₀₀ = ∼3.5). <b>C.</b> Arabinose-inducible expression of the <i>swrW</i> gene in an <i>swrW</i> transposon mutant strain restores hemolysis. <b>D.</b> Swarming motility defect of the <i>swrW</i> mutant is restored by induced expression of the <i>swrW</i> gene.</p

Application of <i>N</i>‑Halogeno‑<i>N</i>‑sodiobenzenesulfonamide Reagents to the Selective Detection of 5‑Methylcytosine in DNA Sequences

To surmount the challenges of the locus determination and accurate quantification of 5-methyl-2′-deoxycytidine (^5MedC) in DNA fragments that contain multiple ^5MedC residues, we designed and synthesized two <i>N</i>-halogeno-<i>N</i>-sodiobenzenesulfonamide reagents that provide a new chemical method for probing ^5MedC in DNA sequences. When the strategy we provided was combined with β-glucosyltransferase, ^5MedC could be distinguished from 5-hydroxymethyl-2′-deoxycytidine (^5hmdC) and deoxycytidine (dC) through the introduction of a glucose moiety to the hydroxyl group of ^5hmdC