A Label-Free Nanogold DNAzyme-Cleaved Surface-Enhanced Resonance Raman Scattering Method for Trace UO2 2+ Using Rhodamine 6G as Probe

In pH?5.5 2-(N-morpholino)-ethanosulfonic acid buffer solution containing 0.25 M NaCl at 80 °C, the single-stranded substrate DNA hybridizes with the enzyme DNA to form double-stranded DNA (dsDNA). The substrate chain of dsDNA could be cracked catalytically by UO₂ ²⁺ to produce a short single-stranded DNA (ssDNA) that adsorbed on the nanogold (NG) surface to form a stable nanogold–ssDNA conjugate and then further combine with rhodamine 6G (RhG) to form a NG–ssDNA–RhG conjugate that can be monitored by the surface-enhanced resonance Raman scattering (SERRS) spectral technique at 1,360 cm^?1. Under the selected conditions, the increased SERRS intensity ΔI ₁₃₆₀ was linear to UO₂ ²⁺ concentration in the range of 5–125 nmol/L, with a detection limit of 1.6 nmol/L. Using a 0.5-μmol/L Hg²⁺ as enhancer, a 2.5–100-nmol/L UO₂ ²⁺ can be determined.     

Characterization of the binding of paylean and DNA by fluorescence,UV spectroscopy and molecular docking techniques

The interaction of paylean (PL) with calf thymus DNA (ctDNA) was investigated using fluorescence spectroscopy, UV absorption, melting studies, ionic strength, viscosity experiments and molecular docking under simulated physiological conditions. Values for the binding constant K_a between PL and DNA were 5.11 × 10³, 2.74 × 10³ and 1.74 × 10³ L mol^–1 at 19, 29 and 39°C respectively. DNA quenched the intrinsic fluorescence of PL via a static quenching procedure as shown from Stern–Volmer plots. The relative viscosity and the melting temperature of DNA were basically unchanged in the presence of PL. The fluorescence intensity of PL–DNA decreased with increasing ionic strength. The value of K_a for PL with double‐stranded DNA (dsDNA) was larger than that for PL with single‐stranded DNA (ssDNA). All the results revealed that the binding mode was groove binding, and molecular docking further indicated that PL was preferentially bonded to A–T‐rich regions of DNA. The values for ΔH, ΔS and ΔG suggested that van der Waals forces or hydrogen bonding might be the main acting forces between PL and DNA. The binding distance was determined to be 3.37 nm based on the theory of Förster energy transference, which indicated that a non‐radiation energy transfer process occurred. Copyright © 2015 John Wiley & Sons, Ltd.     

A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA‐damage repair and DNA replication take place on single‐stranded DNA (ssDNA) or require specific single‐stranded/double‐stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single‐molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single‐molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force‐induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force‐induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.     

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA‐based nanomaterials because of its direct recognition of natural double‐stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single‐strand DNA (ssDNA) fluorescent probes and fluorescent probe–double‐strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K_a) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20‐mer perfectly matched ssDNA probe and three single‐base mismatched ssDNA probes with 146‐mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single‐base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly selective and simple fluorescent sensor for mercury (II) ion detection based on cysteamine‐capped CdTe quantum dots synthesized by the reflux method

Cysteamine (CA)‐capped CdTe quantum dots (QDs) (CA–CdTe QDs) were prepared by the reflux method and utilized as an efficient nano‐sized fluorescent sensor to detect mercury (II) ions (Hg²⁺). Under optimum conditions, the fluorescence quenching effect of CA–CdTe QDs was linear at Hg²⁺ concentrations in the range of 6.0–450 nmol/L. The detection limit was calculated to be 4.0 nmol/L according to the 3σ IUPAC criteria. The influence of 10‐fold Pb²⁺, Cu²⁺ and Ag⁺ on the determination of Hg²⁺ was < 7% (superior to other reports based on crude QDs). Furthermore, the detection sensitivity and selectivity were much improved relative to a sensor based on the CA–CdTe QDs probe, which was prepared using a one‐pot synthetic method. This CA–CdTe QDs sensor system represents a new feasibility to improve the detection performance of a QDs sensor by changing the synthesis method. Copyright © 2014 John Wiley & Sons, Ltd.     

A highly sensitive fluorescence sensor based on lucigenin/chitosan/SiO2 composite nanoparticles for microRNA detection using magnetic separation

In this paper, a convenient reverse‐phase microemulsion method for the synthesis of SiO₂ nanoparticles (NPs) by simply introducing the chitosan and fluorescent dye of lucigenin during the formation reaction of SiO₂ NPs was proposed. Addition of chitosan can make the SiO₂ NPs porous, and increases lucigenin molecule incorporation into chitosan/SiO₂ NPs nanopores based on electrostatic interaction and supermolecular forces. Therefore, fluorescence quantum yield of the lucigenin/chitosan/SiO₂ composite nanoparticles was increased by introduction of chitosan and compared with lucigenin/SiO₂ NPs without chitosan. Because the number of negative charges carried when using single‐stranded DNA (ssDNA) was different from that of double‐stranded DNA (dsDNA), the numbers of lucigenin/chitosan/SiO₂ composite nanoparticles with positive charge adsorbed using ssDNA or dsDNA were different. Consequently, fluorescence intensity caused using ssDNA or dsDNA/miRNA was clearly discriminative. With increase in target DNA/miRNA concentration, the difference in fluorescence intensity also increased, resulting in a good linear relationship between fluorescence intensity sensitizing value and target miRNA concentrations. Therefore, a new fluorescence analysis method for direct detection of let‐7a in human gastric cancer cell samples without enzyme, label free and no immobilization was established using lucigenin/chitosan/SiO₂ composite nanoparticles as a DNA hybrid indicator. The proposed method had high sensitivity and selectivity, low cost and the detection limit was 10 fM (S/N = 3).     

Reactions of 4‐[Bis(2‐chloroethyl)amino]benzenebutanoic Acid (Chlorambucil) with DNA

4‐[Bis(2‐chloroethyl)amino]benzenebutanoic acid (=chlorambucil, 1 ; 2.5 mM ) was allowed to react with single‐ and double‐stranded calf thymus DNA at physiological pH (cacodylic acid, 50% base) at 37°. The DNA–chlorambucil adducts were identified by analyzing the DNA hydrolysates by NMR, UV, HPLC, LC/ESI‐MS/MS techniques as well as by spiking with authentic materials. ssDNA was more reactive than dsDNA, and the order of reactivity in ssDNA was Ade‐N1>Gua‐N7>Cyt‐N3>Ade‐N3. The most reactive site in dsDNA was Ade‐N3. The Gua‐N7 and Ade‐N3 adducts were hydrolytically labile. Ade‐N7 adduct could not be identified in the hydrolysates of ssDNA or dsDNA. The adduct Gua‐N7,N7, which consists of two units of Gua bound together with a unit derived from chlorambucil, is a cross‐linking adduct, and it was detected in the hydrolysates of ssDNA and dsDNA. Also several other adducts were detected which could be characterized by spiking with previously isolated authentic adducts or tentatively by MS. The role of chlorambucil–DNA adducts on the cytotoxicity and mutagenity of 1 is also discussed.     

Surface shapes and surrounding environment analysis of single‐ and double‐stranded DNA‐binding proteins in protein‐DNA interface

Protein‐DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double‐stranded DNA‐binding proteins (DSBs) and single‐stranded DNA‐binding proteins (SSBs) in protein‐DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single‐stranded DNA) or dsDNA (double‐stranded DNA). Proteins 2016; 84:979–989. © 2016 Wiley Periodicals, Inc.     

Study on the interaction between ginsenoside Rh2 and calf thymus DNA by spectroscopic techniques

The interaction between ginsenoside Rh2 (G‐Rh2) and calf thymus DNA (ctDNA) was investigated by spectroscopic methods including UV–vis absorption, fluorescence and circular dichroism (CD) spectroscopy, coupled with DNA melting techniques and viscosity measurements. Stern–Volmer plots at different temperatures proved that the quenching mechanism was a static quenching procedure. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were calculated to be –22.83 KJ · mol^–1and 15.11 J · mol^–1 · K^–1by van ’t Hoff equation, suggesting that hydrophobic force might play a major role in the binding of G‐Rh2 to ctDNA. Moreover, the fluorescence quenching study with potassium iodide as quencher indicated that the K_SV (Stern–Volmer quenching constant) value for the bound G‐Rh2 with ctDNA was lower than the free G‐Rh2. The relative viscosity of ctDNA increased with the addition of G‐Rh2 and also the ctDNA melting temperature increased in the presence of G‐Rh2. Denatured DNA studies showed that quenching by single‐stranded DNA was less than that by double‐stranded DNA. The observed changes in CD spectra also demonstrated that the intensities of the positive and negative bands decreased with the addition of G‐Rh2. The experimental results suggest that G‐Rh2 molecules bind to ctDNA via an intercalative binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

Rhodobacter capsulatus DprA is essential for RecA‐mediated gene transfer agent (RcGTA) recipient capability regulated by quorum‐sensing and the CtrA response regulator

Gene transfer agents (GTAs) are genetic exchange elements that resemble small DNA bacteriophages that transfer random pieces of the producing cell's genome to recipient cells. The best‐studied GTA is that of Rhodobacter capsulatus, termed RcGTA. We discovered that the putative response regulator CtrA, which is essential for RcGTA production, is required for RcGTA‐mediated gene acquisition, and confirmed that a RecA homologue is required. It was also discovered that a DprA (DNA‐protecting protein A) homologue is essential for RcGTA‐mediated gene acquisition, and that dprA expression is induced by gtaI‐dependent quorum‐sensing and non‐phosphorylated CtrA. Modelling of the R. capsulatus DprA structure indicated the presence of a C‐terminal region that resembles a dsDNA‐binding protein domain. Purified His‐tagged R. capsulatus DprA protein bound to both single‐stranded (ss)DNA and double‐stranded (ds)DNA, but with a greater affinity for ssDNA. Additionally, DprA protected dsDNA from endonuclease digestion, and increased the rate of nucleation of Escherichia coli RecA onto ssDNA. Single‐cell expression analyses revealed that dprA is expressed in the majority of cells throughout a population. Overall, the results suggest that incorporation of RcGTA DNA into the recipient cell genome proceeds through a homologous recombination pathway resembling DNA recombination in natural transformation.     

The solution structure of full‐length dodecameric MCM by SANS and molecular modeling

The solution structure of the full‐length DNA helicase minichromosome maintenance protein from Methanothermobacter thermautotrophicus was determined by small‐angle neutron scattering (SANS) data together with all‐atom molecular modeling. The data were fit best with a dodecamer (dimer of hexamers). The 12 monomers were linked together by the B/C domains, and the adenosine triphosphatase (AAA+) catalytic regions were found to be freely movable in the full‐length dodecamer both in the presence and absence of Mg²⁺ and 50‐meric single‐stranded DNA (ssDNA). In particular, the SANS data and molecular modeling indicate that all 12 AAA+ domains in the dodecamer lie approximately the same distance from the axis of the molecule, but the positions of the helix–turn–helix region at the C‐terminus of each monomer differ. In addition, the A domain at the N‐terminus of each monomer is tucked up next to the AAA+ domain for all 12 monomers of the dodecamer. Finally, binding of ssDNA does not lock the AAA+ domains in any specific position, which leaves them with the flexibility to move both for helicase function and for binding along the ssDNA. Proteins 2014; 82:2364–2374. © 2014 Wiley Periodicals, Inc.     

Purification of glucose‐6‐phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme on which the pentose phosphate pathway was checked. In this study, purification of a G6PD enzyme was carried out by using rat erythrocytes with a specific activity of 13.7 EU/mg and a yield of 67.7 and 155.6‐fold by using 2′,5′‐ADP Sepharose‐4B affinity column chromatography. For the purpose of identifying the purity of enzyme and molecular mass of the subunit, a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was carried out. The molecular mass of subunit was calculated 56.5 kDa approximately. Then, an investigation was carried out regarding the inhibitory effects caused by various metal ions (Fe²⁺, Pb²⁺, Cd²⁺, Ag⁺, and Zn²⁺) on G6PD enzyme activities, as per Beutler method at 340 nm under in vitro conditions. Lineweaver–Burk diagrams were used for estimation of the IC₅₀ and K_i values for the metals. K_i values for Pb⁺², Cd⁺², Ag⁺, and Zn⁺² were 113.3, 215.2, 19.4, and 474.7 μM, respectively.     

A N‐(2‐hydroxyethyl)piperazine dangled 2,5‐diphenyl‐1,3,4‐oxadiazole‐based fluorescent sensor for selective relay recognition of Cu2+ and sulfide in water

A new 2,5‐diphenyl‐1,3,4‐oxadiazole‐based derivative (L) was synthesized and applied as a highly selective and sensitive fluorescent sensor for relay recognition of Cu²⁺ and S^2? in water (Tris–HCl 10 mM, pH = 7.0) solution. L exhibits an excellent selectivity to Cu²⁺ over other examined metal ions with a prominent fluorescence ‘turn‐off’ at 392 nm. L interacts with Cu²⁺ through a 1:2 binding stoichiometry with a detection limit of 4.8 × 10^–7 M. The on‐site formed L–2Cu²⁺ complex exhibits excellent selectivity to S^2? with a fluorescence ‘off–on’ response via a Cu²⁺ displacement approach. Copyright © 2016 John Wiley & Sons, Ltd.     

Micronomicin/tobramycin binding with DNA: fluorescence studies using of ethidium bromide as a probe and molecular docking analysis

Two aminoglycosides, micronomicin (MN), and tobramycin (TB), binding with DNA were studied using various spectroscopic techniques including fluorescence, UV–Vis, FT-IR, and CD spectroscopy coupled with relative viscosity and molecular docking. Studies of fluorescence quenching and time-resolved fluorescence spectroscopy all revealed that MN/TB quenching the fluorescence of DNA–EB belonged to static quenching. The binding constants and binding sites were obtained. The values of ΔH, ΔS, and ΔG suggested that van der Waals force or hydrogen bond might be the main binding force. FT-IR and CD spectroscopy revealed that the binding of MN/TB with DNA had an effect on the secondary structure of DNA. Binding mode of MN/TB with DNA was groove binding which was ascertained by viscosity measurements, CD spectroscopy, ionic strength, melting temperature (T_m), contrast experiments with single stranded (ssDNA), and double stranded DNA (dsDNA). Molecular docking analysis further confirmed that the groove binding was more acceptable result.     

5′‐Single‐stranded/duplex DNA junctions are loading sites for E. coli UvrD translocase

Escherichia coli UvrD is a 3′–5′ superfamily 1A helicase/translocase involved in a variety of DNA metabolic processes. UvrD can function either as a helicase or only as an single‐stranded DNA (ssDNA) translocase. The switch between these activities is controlled in vitro by the UvrD oligomeric state; a monomer has ssDNA translocase activity, whereas at least a dimer is needed for helicase activity. Although a 3′‐ssDNA partial duplex provides a high‐affinity site for a UvrD monomer, here we show that a monomer also binds with specificity to DNA junctions possessing a 5′‐ssDNA flanking region and can initiate translocation from this site. Thus, a 5′‐ss–duplex DNA junction can serve as a high‐affinity loading site for the monomeric UvrD translocase, whereas a 3′‐ss–duplex DNA junction inhibits both translocase and helicase activity of the UvrD monomer. Furthermore, the 2B subdomain of UvrD is important for this junction specificity. This highlights a separation of helicase and translocase function for UvrD and suggests that a monomeric UvrD translocase can be loaded at a 5′‐ssDNA junction when translocation activity alone is needed.     

Terbium (III) coordination polymer–copper (II) compound as fluorescent probe for time‐resolved fluorescence ‘turn‐on’ detection of hydrogen sulfide

With recognition of the biological importance of hydrogen sulfide (H₂S), we present a simple and effective fluorescent probe for H₂S using a Tb³⁺ coordination polymer–Cu²⁺ compound (DPA/Tb/G–Cu²⁺). Dipicolinic acid (DPA) and guanosine (G) can coordinate with Tb³⁺ to form a macromolecular coordination polymer (DPA/Tb/G). DPA/Tb/G specifically binds to Cu²⁺ in the presence of coexisting cations, and obvious fluorescence quenching is observed. The quenched fluorescence can be exclusively recovered upon the addition of sulfide, which is measured in the mode of time‐resolved fluorescence. The fluorescence intensities of the DPA/Tb/G–Cu²⁺ compound enhance linearly with increasing sulfide concentrations from 1 to 30 μM. The detection limit for sulfide in aqueous solution is estimated to be 0.3 μM (at 3σ). The DPA/Tb/G–Cu²⁺ compound was successfully applied to sense H₂S in human serum samples and exhibited a satisfactory result. It displays some desirable properties, such as fast detection procedure, high selectivity and excellent sensitivity. This method is very promising to be utilized for practical detection of H₂S in biological and environmental samples.     

A tricorn‐rhodamine fluorescent chemosensor for detection of Co2+ ions

A novel chemosensor TrisRh based on tris(2‐aminoethyl)amine and rhodamine 6G is designed and synthesized as a fluorescence turn‐on probe for Co²⁺ ions that is paramagnetic with a property of quenching fluorescence. Rhodamine spirolactam forms are nonfluorescent, whereas, ring‐opening of corresponding spirocyclic induced by Co²⁺ results in strong fluorescence emission. Upon the addition of Co²⁺ ions, TrisRh can display significant enhancements in absorbance and fluorescence intensity as well as evident colorific transformation, which can be perceived by the naked eye. The association stoichiometry of TrisRh to Co²⁺ ions was inferred to be 1:1 through Job's plot and electrospray ionization mass spectrometry analysis. The binding model was speculated from Fourier transform infrared spectra and ¹H–nuclear magnetic resonance technologies. Significantly, the limit of detection was determined to be as low as 1.22 nmol. Furthermore, TrisRh can exhibit robust anti‐jamming ability against other interference metal ions.