Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells.

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.

Visualizing ribonuclease digestion of RNA-like polymers produced by hot wet-dry cycles.

Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.

Silver Nanoclusters Serve as Fluorescent Rivets Linking Hoogsteen Triplex DNA and Hairpin-Loop DNA Structures.

Greater understanding of the mutual influence between DNA and the associated nanomaterial on the properties of each other can provide alternative strategies for designing and developing DNA nanomachines. DNA secondary structures are essential for encapsulating highly emissive silver nanoclusters (DNA/AgNCs). Likewise, AgNCs stabilize secondary DNA structures, such as hairpin DNA, duplex DNA, and parallel-motif DNA triplex. In this study, we found that the fluorescence of AgNCs encapsulated within a Hoogsteen triplex DNA structure can be turned on and off in response to pH changes. We also show that AgNCs can act as nanoscale rivets, linking two functionally distinctive DNA nanostructures. For instance, we found that a Hoogsteen triplex DNA structure with a seven-cytosine loop encapsulates red fluorescent AgNCs. The red fluorescence faded under alkaline conditions, whereas the fluorescence was restored in a near-neutral environment. Hairpin DNA and random DNA structures did not exhibit this pH-dependent AgNCs fluorescence. A fluorescence lifetime measurement and a small-angle X-ray scattering analysis showed that the triplex DNA-encapsulated AgNCs were photophysically convertible between bright and dark states. An in-gel electrophoresis analysis indicated that bright and dark convertibility depended on the AgNCs-riveted dimerization of the triplex DNAs. Moreover, we found that AgNCs rivet the triplex DNA and hairpin DNA to form a heterodimer, emitting orange fluorescence. Our findings suggest that AgNCs between two cytosine-rich loops can be used as nanorivets in designing noncanonical DNA origami beyond Watson-Crick base pairing.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Detoxification of toxin A and toxin B by copper ion-catalyzed oxidation in production of a toxoid-based vaccine against Clostridioides difficile.

Clostridioides difficile infections (CDI) has emerged worldwide as a serious antimicrobial-resistant healthcare-associated disease resulting in diarrhea and pseudomembranous colitis. The two cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB) are the major virulence factor responsible for the disease symptoms. We examined time-dependent oxidative detoxification of TcdA and TcdB using different molar ratios of protein:Cu2+:H2O2. The metal-catalyzed oxidation (MCO) reaction in molar ratios of 1:60:1000 for protein:Cu2+:H2O2 at pH 4.5 resulted in a significant 6 log10 fold reduction in cytotoxicity after 120-min incubation at 37 °C. Circular dichroism revealed that MCO-detoxified TcdA and TcdB had secondary and tertiary structural folds similar to the native proteins. The conservation of immunogenic epitopes of both proteins was tested using monoclonal antibodies in an ELISA, comparing our MCO-detoxification approach to a conventional formaldehyde-detoxification method. The oxidative detoxification of TcdA and TcdB led to an average 2-fold reduction in antibody binding relative to native proteins, whereas formaldehyde cross-linking resulted in 3-fold and 5-fold reductions, respectively. Finally, we show that mice immunized with a vaccine consisting of MCO-detoxified TcdA and TcdB were fully protected against disease symptoms and death following a C. difficile infection and elicited substantial serum IgG responses against both TcdA and TcdB. The results of this study present copper ion-catalyzed oxidative detoxification of toxic proteins as a method highly suitable for the rapid production of safe, immunogenic and irreversible toxoid antigens for future vaccine development and may have the potential for replacing cross-linking reagents like formaldehyde.

Noncanonical Head-to-Head Hairpin DNA Dimerization Is Essential for the Synthesis of Orange Emissive Silver Nanoclusters.

DNA secondary structures, such as dimers and hairpins, are important for the synthesis of DNA template-embedded silver nanoclusters (DNA/AgNCs). However, the arrangement of AgNCs within a given DNA template and how the AgNC influences the secondary structure of the DNA template are still unclear. Here, we introduce a noncanonical head-to-head hairpin DNA nanostructure that is driven by orange-emissive AgNCs. Through detailed in-gel analysis, sugar backbone switching, inductively coupled plasma mass spectrometry, small-angle X-ray scattering, and small angle neutron scattering, we show that the orange-emissive AgNCs mediate cytosine-Ag-cytosine bridging between two six-cytosine loop (6C-loop) hairpin DNA templates. Unlike green, red, or far-red emissive AgNCs, which are embedded inside a hairpin and duplex DNA template, the orange-emissive AgNCs are localized on the interface between the two 6C-loop hairpin DNA templates, thereby linking them. Moreover, we found that deoxyribose in the backbone of the 6C-loop at the third and fourth cytosines is crucial for the formation of the orange-emissive AgNCs and the head-to-head hairpin DNA structure. Taken together, we suggest that the specific wavelength of AgNCs fluorescence is determined by the mutual interaction between the secondary or tertiary structures of DNA- and AgNC-mediated intermolecular DNA cross-linking.

The structural shift of a DNA template between a hairpin and a dimer tunes the emission color of DNA-templated AgNCs.

The scaffolding DNA sequence and the size of silver nanoclusters (AgNCs), confined in a DNA template are the key parameters in determining the fluorescent properties of DNA-stabilized silver nanoclusters (DNA/AgNCs). In addition, we suggest here that the structural shift of a DNA hairpin-dimer is as important as the DNA sequence in determining the emission wavelength of DNA/AgNCs. Furthermore, we show that the structural shift post AgNC formation can be triggered by incubation time and pre-AgNC formation under salt conditions. As an important factor in predicting the emission properties of DNA/AgNCs, the modulation of DNA secondary structures with either sequence changes or ionic conditions can be applied for the dual-color detection system of a target molecule. Particularly, the dual-color detection method may increase the reliability of DNA/AgNC sensors for miRNAs.

MicroRNA Biomarkers in Neurodegenerative Diseases and Emerging Nano-Sensors Technology.

MicroRNAs (miRNAs) are essential small RNA molecules (20-24 nt) that negatively regulate the expression of target genes at the post-transcriptional level. Due to their roles in a variety of biological processes, the aberrant expression profiles of miRNAs have been identified as biomarkers for many diseases, such as cancer, diabetes, cardiovascular disease and neurodegenerative diseases. In order to precisely, rapidly and economically monitor the expression of miRNAs, many cutting-edge nanotechnologies have been developed. One of the nanotechnologies, based on DNA encapsulated silver nanoclusters (DNA/AgNCs), has increasingly been adopted to create nanoscale bio-sensing systems due to its attractive optical properties, such as brightness, tuneable emission wavelengths and photostability. Using the DNA/AgNCs sensor methods, the presence of miRNAs can be detected simply by monitoring the fluorescence alteration of DNA/AgNCs sensors. We introduce these DNA/ AgNCs sensor methods and discuss their possible applications for detecting miRNA biomarkers in neurodegenerative diseases.

Electrochemical reduction of nitroaromatic compounds by single sheet iron oxide coated electrodes.

Nitroaromatic compounds are substantial hazard to the environment and to the supply of clean drinking water. We report here the successful reduction of nitroaromatic compounds by use of iron oxide coated electrodes, and demonstrate that single sheet iron oxides formed from layered iron(II)-iron(III) hydroxides have unusual electrocatalytic reactivity. Electrodes were produced by coating of single sheet iron oxides on indium tin oxide electrodes. A reduction current density of 10 to 30µAcm(-2) was observed in stirred aqueous solution at pH 7 with concentrations of 25 to 400µM of the nitroaromatic compound at a potential of -0.7V vs. SHE. Fast mass transfer favors the initial reduction of the nitroaromatic compound which is well explained by a diffusion layer model. Reduction was found to comprise two consecutive reactions: a fast four-electron first-order reduction of the nitro-group to the hydroxylamine-intermediate (rate constant=0.28h(-1)) followed by a slower two-electron zero-order reduction resulting in the final amino product (rate constant=6.9µM h(-1)). The zero-order of the latter reduction was attributed to saturation of the electrode surface with hydroxylamine-intermediates which have a more negative half-wave potential than the parent compound. For reduction of nitroaromatic compounds, the SSI electrode is found superior to metal electrodes due to low cost and high stability, and superior to carbon-based electrodes in terms of high coulombic efficiency and low over potential.

Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs.

MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.

DNA/RNA chimera templates improve the emission intensity and target the accessibility of silver nanocluster-based sensors for human microRNA detection.

In recent years microRNAs (miRNAs) have been established as important biomarkers in a variety of diseases including cancer, diabetes, cardiovascular disease, aging, Alzheimer's disease, asthma, autoimmune disease and liver diseases. As a consequence, a variety of monitoring methods for miRNAs have been developed, including a fast and simple method for miRNA detection by exploitation of the unique photoluminescence of DNA-templated silver nanoclusters (DNA/AgNCs). To increase the versatility of the AgNC-based method, we have adopted DNA/RNA chimera templates for AgNC-based probes, allowing response from several human miRNAs which are hardly detectable with DNA-based probes. Here, we demonstrate in detail the power of DNA/RNA chimera/AgNC probes in detecting two human miRNAs, let-7a and miR-200c. The DNA/RNA chimera-based probes are highly efficient to determine the level of miRNAs in several human cell lines.

DNA-RNA chimera indicates the flexibility of the backbone influences the encapsulation of fluorescent AgNC emitters.

Many DNA scaffolds efficiently encapsulate highly emissive silver nanoclusters (AgNCs). The secondary structures and the arrangement of sequences of DNA scaffolds are important factors by which the specific features of AgNCs emitters can be determined. By introducing DNA-RNA chimera scaffolds, we here explore another factor - the flexibility of the backbone of nucleic acid-templates - in creating highly fluorescent AgNC emitters.

Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein from Clostridium difficile.

Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging. This work was aimed at the structural characterization of a newly identified Fic protein of the Gram-positive bacterium Clostridium difficile. A constitutively active inhibitory helix mutant of C. difficile Fic was overexpressed in Escherichia coli, purified and crystallized by the vapour-diffusion technique. Preliminary X-ray crystallographic analysis shows that the crystals diffract to at least 1.68 Šresolution at a synchrotron X-ray source. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a=45.6, b=80.8, c=144.7 Å, α=ß=γ=90°. Two molecules per asymmetric unit corresponds to a Matthews coefficient of 2.37 Å3 Da(-1) and a solvent content of 48%.

In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes.

MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method.

An L-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of D-sorbose with enzymatic or electrochemical cofactor regeneration.

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an L-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with D-glucitol oxidation to D-fructose but also converted L-glucitol to D-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K m value for L-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a D-sorbitol dehydrogenase (EC 1.1.1.14).

Nonribosomal peptide synthetase (NPS) genes in Fusarium graminearum, F. culmorum and F. pseudograminearium and identification of NPS2 as the producer of ferricrocin.

Fungi have the potential to produce a wide range of secondary metabolites including polyketides and small peptides produced by nonribosomal peptide synthetases (NPS). Fusarium graminearum is a mycotoxin producing pathogen of cereals and knowledge of the infection process is essential for the development of disease control. Bioinformatics provide a means to identify genes encoding NPSs, the products of which may act as fungal virulence factors. The F. graminearum genome sequence was analysed and similarity searches and application of prediction server service identified 15 putative NPS genes. NPS1 and NPS2, were found to be related to genes involved in NPS hydroxamate siderophore biosynthesis and chemical analysis of a F. graminearum NPS2 deletion mutant showed that this gene encodes the NPS responsible for the biosynthesis of ferricrocin. The expression of the NPS genes was analysed in Fusarium culmorum. NPS1 and NPS19 differed from the remainder of the genes, as they were only expressed during infection of barley roots and not under the different culture conditions tested. Strains of F. graminearum, F. culmorum and Fusarium pseudograminearum were examined for the presence and expression of the 15 identified NPS genes. With the exception of NPS18, that is absent in F. pseudograminearum, all the NPS genes are represented in the diffferent species. Lack of transcripts from some genes and the presence of frameshift and stop codons in four of the NPS genes in the sequenced F. graminearum strain suggest that some are pseudogenes.