Dynamics of Fluorescent Dyes Attached to G‐Quadruplex DNA and their Effect on FRET Experiments

FRET spectroscopy is a promising approach for investigating the dynamics of G‐quadruplex DNA folds and improving the targeting of G‐quadruplexes by potential anticancer compounds. To better interpret such experiments, classical and replica‐exchange molecular dynamics simulations and fluorescence‐lifetime measurements are used to understand the behavior of a range of Cy3‐based dyes attached to the 3′ end of G‐quadruplex DNA. The simulations revealed that the dyes interact extensively with the G‐quadruplex. Identification of preferred dye positions relative to the G‐quadruplex in the simulations allows the impact of dye–DNA interactions on FRET results to be determined. All the dyes show significant deviations from the common approximation of being freely rotating and not interacting with the host, but one of the Cy3 dye analogues is slightly closer to this case.     

Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Förster distances for various dye-DNA conjugates

Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The F?rster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been determined. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the F?rster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technology is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity experiments. The differential quenching of TAMRA fluorescence by IabRQ in solution has been used to discriminate between 0 and 3 base pair mismatches at 60 degrees C for a 19 base sequence. At room temperature, the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temperature which occurs beyond two base pair mismatches and reverses the observed trend in TAMRA fluorescence prior to that degree of mismatch. When this experimental system is transferred to a glass surface through covalent coupling and organosilane chemistry, the observed trend in TAMRA fluorescence at room temperature is similar to that obtained in bulk solution, but without a threshold of duplex stability. In addition to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface.     

A theoretical study of reactivity and regioselectivity in the hydroxylation of adamantane by ferrate(VI)

The conversion of adamantane to adamantanols mediated by ferrate (FeO(4)(2)(-)), monoprotonated ferrate (HFeO(4)(-)), and diprotonated ferrate (H(2)FeO(4)) is discussed with the hybrid B3LYP density functional theory (DFT) method. Diprotonated ferrate is the best mediator for the activation of the C-H bonds of adamantane via two reaction pathways, in which 1-adamantanol is formed by the abstraction of a tertiary hydrogen atom (3 degrees ) and 2-adamantanol by the abstraction of a secondary hydrogen atom (2 degrees ). Each reaction pathway is initiated by a C-H bond cleavage via an H-atom abstraction that leads to a radical intermediate, followed by a C-O bond formation via an oxygen rebound step to lead to an adamantanol complex. The activation energies for the C-H cleavage step are 6.9 kcal/mol in the 1-adamantanol pathway and 8.4 kcal/mol in the 2-adamantanol pathway, respectively, at the B3LYP/6-311++G level of theory, whereas those of the second reaction step corresponding to the rebound step are relatively small. Thus, the rate-determining step in the two pathways is the C-H bond dissociation step, which is relevant to the regioselectivity for adamantane hydroxylation. The relative rate constant (3 degrees )/(2 degrees ) for the competing H-atom abstraction reactions is calculated to be 9.30 at 75 degrees C, which is fully consistent with an experimental value of 10.1.     

The photoluminescent graphene oxide serves as an acceptor rather than a donor in the fluorescence resonance energy transfer pair of Cy3.5-graphene oxide

We have systematically studied the fluorescence resonance energy transfer (FRET) efficiency between the photoluminescent graphene oxide (GO) and Cy3.5 dye by controlling the donor-acceptor distance with a double stranded DNA and demonstrated that the GO serves as an acceptor rather than a donor in this FRET system.     

Can luminescent quantum dots be efficient energy acceptors with organic dye donors?

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.     

Impact of ligand protonation on higher-order metal complexation kinetics in aqueous systems

The impact of ligand protonation on the complexation kinetics of higher-order complexes is quantitatively described. The theory is formulated on the basis of the usual situation for metal complex formation in aqueous systems in which the exchange of water for the ligand in the inner coordination sphere is rate-determining (Eigen mechanism). We derive expressions for the general case of lability of ML(n) species that account for the contributions from all outer-sphere complexes to the rate of complex formation. For dynamic complexes, dissociation of ML is usually the rate-determining step in the overall process ML(n) --> M. Under such conditions, it is the role of ligand protonation in the step ML --> M that is relevant for the kinetic flux. 1:2 complexes of Cd(II) with pyridine-2,6-dicarboxylic acid fall into this category, and their lability at a microelectrode is reasonably well predicted by the differentiated approach. For non-dynamic systems, the kinetic flux arising from dissociation of higher-order complexes contributes to the rate-determining step. In this case, the weighted contribution of protonated and unprotonated outer-sphere complexes in all contributing dissociation reactions must be taken into account. The kinetic flux arising from the dissociation of 1:2 complexes of Ni(II) with bicine at a conventional electrode was quite well described by this combined approach. The results establish the generic role of ligand protonation within the overall framework of metal complexation kinetics in which complexes may be dynamic to an extent that depends on the operational time scale of the measurement technique.     

Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb(3+) fluorescent chelate with N,N,N('),N(')-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb(3+)) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb(3+) labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb(3+) labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ngml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement.     

Ni催化剂上一氧化碳加氢反应机理研究

Ｎｉ催化剂上一氧化碳加氢反应机理研究胡云行，万惠霖，关玉德，林恒生（厦门大学化学系，固体表面物理化学国家重点实验室，厦门，３６１００５）（中国科学院山西煤炭化学研究所，太原）关键词脉冲法，一氧化碳，加氢，反应机理自本世纪初报道了一氧化碳加氢生成甲烷以...     

Hierarchical Unfolding of Mj HSP16.5

Mj HSP16.5 is a small heat shock protein (sHSP) from the hyperthermophilic methanoarchaeon, Methanococcus jannaschii (Mj), which lives at the environment of high temperature up to 94 °C. The structural data showed that Mj HSP16.5 was a 24-mer that formed a hollow sphere with octahedral symmetry. Mj HSP16.5 was very stable at pH 7 that it maintained the 24-mer structure even at 85 °C. In the present study, we investigated the unfolding process of Mj HSP16.5 in the presence of denaturants using several techniques, including circular dichroism (CD), dynamic light scattering (DLS), fluorescence spectroscopy, and size exclusive chromatography (SEC). We found that 8 mol·L⁻¹ urea had no obvious effect on the structure of Mj HSP16.5 at pH 7. The unfolding of Mj HSP16.5 at pH 7 in the presence of guanidine hydrochloride (GdHCl) showed hierarchical behavior. Three significant transitions were observed around 2.0, 3.0, and 6.0 mol·L⁻¹ GdHCl at pH 7. ANS (8-anilino-1- naphthalenesulfonic acid) titration results showed that the binding ability of Mj HSP16.5 to ANS decreased gradually as the concentration of GdHCl increased until around 2.0 mol·L⁻¹ GdHCl, indicating surface hydrophobic area change, and this first transition was companioned with precipitation of Mj HSP16.5. Acrylamide quenching of fluorescence showed that the Stern-Volmer constant changed at about 3.0 mol·L⁻¹ GdHCl, indicating changes of the dimeric interface, and this phase transition was companioned with oligomeric state change from 24-mer to small oligomers (4-mer to 8-mer). The last unfolding phase started around 5.0 mol·L⁻¹ GdHCl, with a midpoint of 6.1 mol·L⁻¹ GdHCl, and Mj HSP16.5 was completely unfolded at 7.0 mol·L⁻¹ GdHCl. We also found that Mj HSP16.5 could be quite easily unfolded at pH 3, where it could be completely unfolded in 4.0 mol·L⁻¹ GdHCl.     

Verification and Biophysical Characterization of a New Three‐Color Förster Resonance‐Energy‐Transfer (FRET) System in DNA

We report on a new three‐color FRET system consisting of three fluorescent dyes, i.e., of a carbostyril (=quinolin‐2(1H)‐one)‐derived donor D, a (bathophenanthroline)ruthenium complex as a relay chromophore A₁, and a Cy dye as A₂ (FRET=Förster resonance‐energy‐transfer) (cf. Fig. 1). With their widely matching spectroscopic properties (cf. Fig. 2), the combination of these dyes yielded excellent FRET efficiencies. Furthermore, fluorescence lifetime measurements revealed that the long fluorescence lifetime of the Ru complex was transferred to the Cy dye offering the possibility to measure the whole system in a time‐resolved mode. The FRET system was established on double‐stranded DNA (cf. Fig. 3) but it should also be generally applicable to other biomolecules.     

小分子热休克蛋白Mj HSP16.5的分级变性

应用荧光光谱、圆二色光谱、体积排阻色谱、激光动态光散射等技术, 研究了来自嗜热古细菌Methanococcus jannaschii (Mj)的小分子热休克蛋白Mj HSP16.5在变性剂作用下的变性过程. 研究表明, 在pH 7时, Mj HSP16.5在8 mol·L-1尿素作用下不会发生变性. 在pH 7条件下, 盐酸胍对Mj HSP16.5的变性表现为一个分级过程,分别在2.0、3.0和6.0 mol·L-1盐酸胍浓度附近,出现明显的结构变化; 到7.0 mol·L-1盐酸胍时, Mj HSP16.5才完全变性. 降低溶液pH值将使Mj HSP16.5的变性变得更为容易.     

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA).     

Multiple isotope effect study of the acid-catalyzed hydrolysis of formamide

Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 degrees C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one tetrahedral intermediate, which does not appreciably exchange its carbonyl oxygen with the solvent (kh/kex = 55). The pKa for formamide was determined by 15N NMR and found to be about -2.0. The formyl-hydrogen kinetic isotope effect (KIE) is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl-carbon KIE (13kobs = 1.031) is in agreement with this conclusion. The small leaving-nitrogen KIE (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl-oxygen KIE (18kobs = 0.996) points to attack of water as the rate-determining step. On the basis of these results, a mechanism is proposed in which attachment of the nucleophile to a protonated formamide molecule is rate determining.     

Enantioselective noncovalent synthesis of hydrogen-bonded double-rosette assemblies

The noncovalent synthesis of enantiomerically pure hydrogen-bonded assemblies (M)- and (P)-1(3).(CA)(6) is described. These dynamic assemblies are of one single handedness (M or P), but do not contain any chiral components. They are prepared by using the "chiral memory" concept: the induction of supramolecular chirality is achieved through initial assembly with chiral barbiturates, which are subsequently replaced by achiral cyanurates. This exchange process occurs quantitatively and without loss of the M or P handedness of the assemblies. Racemization studies have been used to determine an activation energy for racemization of 105.9+/-6.4 kJ mol(-1) and a half-life time to racemization of 4.5 days in benzene at 18 degrees C. Kinetic studies have provided strong evidence that the rate-determining step in the racemization process is the dissociation of the first dimelamine component 1 from the assembly 1(3).(CA)(6). In addition to this, it was found that the expelled chiral barbiturate (RBAR or SBAR) acts as a catalyst in the racemization process. Blocking the dissociation process of dimelamines 1 from assembly 1(3).(CA)(6) by covalent capture through a ring-closing metathesis (RCM) reaction produces an increase of more than two orders of magnitude in the half-life time to racemization.     

Lithium-alkyl, -aryl, and -amido ligand-exchange dynamics of dianionic zirconium(IV) complexes with chelating amidophenolate ligands

The synthesis, characterization, and solution behavior of a series of six-coordinate zirconium(IV) dianions [ZrX2(ap)2]2- (ap = 2,4-di-tert-butyl-6-(tert-butylamido)phenolate; X = Ph, 3a; X = p-tolyl, 3b; X = Me, 4; X = NMe2, 5) are described. Complexes 3-5 were prepared by treating the neutral zirconium complex Zr(ap)2(THF)2 (1) with 2 equiv of LiX or by the direct reaction of apLi2 and LiX with ZrCl4. The complexes were isolated as lithium-etherate salts, and they were characterized by NMR spectroscopy and single-crystal X-ray diffraction. In non-coordinating solvents such as benzene-d6, complexes 3-5 are robust in solution, but in coordinating solvents such as THF-d8, dissociation of LiX was observed. The rate of LiX loss was evaluated by exchange reactions; the reaction rate constants span nine orders of magnitude at 298 K, with the slowest reaction being the dissociation of PhLi from 3a (tau1/2 = 4 h) and the fastest reaction being the dissociation of LiNMe2 from 5 (tau1/2 = 53 mus). In the case of LiNMe2 dissociation from 5, activation parameters suggest that the rate-determining step is purely dissociative; however, for diphenyl and dimethyl complexes 3a and 4, respectively, activation parameters suggest a solvent-assisted rate-determining step.     

Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5-mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.     

On the reaction of nickel(II) with thiocyanate ion and other unidentate ligands in dimethylsulfoxide as solvent:I d orD mechanism?

Rate constants and activation parameters for the formation and the dissociation of the monocomplex of nickel(II) with thiocyanate ion in dimethylsulfoxide have been determined by stopped-flow measurements. The results for thiocyanate ion, and also those for several other unidentate ligands, are consistent with a dissociative interchange mechanism in which solvent exchange constitutes the rate-determining step. On the other hand, previous results have shown that for multidentate ligands the ring-closure step also may be rate limiting.     

Donor-conformation-dependent energy transfer for dual-color fluorescent probe with high-resolution imaging

Herein, we presented a brand-new concept to construct the Forster resonance energy transfer(FRET) based cassette by integrating a vibration-induced emission(VIE) chromophore as the donor. Different from traditional donors only with a single emission, the VIE donor possessed well-separated dual emission bands by altering the excited state molecular configuration from the bent state to the planar state. By linking an acceptor such as a cyanine dye(Cy5), a novel VIE-FRET cassette(PPCy5) was prepared. The planar emission profile of the VIE donor moiety could fully cover the absorption of Cy5, and thus the complete FRET process enabled the excellent bimodal spectra difference of 142 nm and ultra-large pseudo-Stokes shift of up to 300 nm.Benefiting from the viscosity-dependent characteristic of the VIE donor, PPCy5 could clearly and intuitively reveal the different viscosity regions in vivo by dual-color and high-resolution imaging. The VIE-FRET paradigm provides an optional platform for developing donor-acceptor-based dual-color fluorescent probes with high-resolution imaging ability.     

Optimization of modification condition of nano-kaoline as an adsorbent for textile dye removal

In this study, adsorption capacity of textile red dye on alkylated kaolin was investigated through batch mode. Accordingly, raw kaolin was alkylated via NaOH treatment. The work was carried out in two steps. At first step, the effect of various alkylation conditions of kaolin on its dye adsorption performance was studied using the model equation designed by 2-level factorial design. Three factors were changed in two level including NaOH solution temperature (45–75°C), mixing time (3–24 h), and NaOH solution concentration (0.1–2 M). The resultant model showed 91% of the variability in data used to fit dye adsorption capacity values. However, the analysis of variance revealed that the fitted model is high significant. Based on the predicting model, the optimal alkylation conditions with desirability factor of 0.911 were obtained at temperature of 75°C, NaOH concentration of 0.1 M and after 24 h mixing. At step two, chemical content, bonds and functional groups of the treated kaolin, which was prepared based on the optimum condition and compared with the raw kaolin via X-ray fluorescence (XRF) analysis and Fourier transform infrared analysis (FTIR). The results show slight reduction in SiO₂ content. Finally, the adsorption capacity of dye on both treated and raw kaolin was investigated.     

Local heating of discrete droplets using magnetic porous silicon-based photonic crystals

This paper describes a method for local heating of discrete microliter-scale liquid droplets. The droplets are covered with magnetic porous Si microparticles, and heating is achieved by application of an external alternating electromagnetic field. The magnetic porous Si microparticles consist of two layers. The top layer contains a photonic code and it is hydrophobic, with surface-grafted dodecyl moieties. The bottom layer consists of a hydrophilic silicon oxide host layer that is infused with Fe3O4 nanoparticles. The amphiphilic microparticles spontaneously align at the interface of a water droplet immersed in mineral oil, allowing manipulation of the droplets by application of a magnetic field. Application of an oscillating magnetic field (338 kHz, 18 A rms current in a coil surrounding the experiment) generates heat in the superparamagnetic particles that can raise the temperature of the enclosed water droplet to >80 degrees C within 5 min. A simple microfluidics application is demonstrated: combining complementary DNA strands contained in separate droplets and then thermally inducing dehybridization of the conjugate. The complementary oligonucleotides were conjugated with the cyanine dye fluorophores Cy3 and Cy5 to quantify the melting/rebinding reaction by fluorescence resonance energy transfer (FRET). The magnetic porous Si microparticles were prepared as photonic crystals, containing spectral codes that allowed the identification of the droplets by reflectivity spectroscopy. The technique demonstrates the feasibility of tagging, manipulating, and heating small volumes of liquids without the use of conventional microfluidic channel and heating systems.