The separation of cyclic diadenosine diphosphorothioate and the diastereomers of its difluorinated derivative and the estimation of the binding constants and ionic mobilities of their complexes with 2-hydroxypropyl-ß-cyclodextrin by affinity capillary electrophoresis.

The incorporation of phosphorothioate linkages has recently been extensively employed in therapeutic oligonucleotides. For their separation and quality control, new high-efficient and high-sensitive analytical methods are needed. In this work, a new affinity capillary electrophoresis method has been developed and applied for the separation of a potential anticancer drug, 2',3'-cyclic diadenosine diphosphorothioate (Rp, Rp) (ADU-S100), and three recently newly synthesized diastereomers of its difluorinated derivative, 3',3'-cyclic di(2'-fluoro, 2'-deoxyadenosine phosphorothioate). The separation was performed in the various background electrolytes (BGEs) within a pH range 5-9 using several native and derivatized cyclodextrins (CDs) as chiral additives of the BGE. Relatively good separations were obtained with ß-, γ-, and 2-hydroxypropyl-γ-CDs in some of the BGEs tested. However, the best separation was achieved using the 2-hydroxypropyl-ß-CD chiral selector at 43.5 mM average concentration in the BGE composed of 40 mM Tris, 40 mM tricine, pH 8.1. Under these conditions, all the previous four cyclic dinucleotides (CDNs) were baseline separated within 4 min. Additionally, the average apparent binding constants and the average actual ionic mobilities of the complexes of all four CDNs with 2-hydroxypropyl-ß-CD in the above BGE were determined. The formed complexes were found to be relatively weak, with the average apparent binding constants in the range of 12.2-94.1 L mol-1 and with the actual ionic mobilities spanning the interval (-7.8 to -12.7) × 10-9 m2 V-1 s-1. The developed method can be applied for the separation, analysis, and characterization of the above and similar CDNs.

Steady State and Time-Resolved Fluorescence Spectroscopy of Cinchonine Dication in Sodium Dodecylsulphate Micellar System.

This paper reports the influence of surface charge of the micelles on to the photophysical properties of a cinchonine dication (C2+) fluorophore in anionic, sodium dodecylsulphate (SDS), surfactant at premicellar, micellar and post-micellar concentrations in aqueous phase at room temperature. The magnitude of edge excitation red shift (EERS) in the fluorescence maximum of C2+ in bulk water solution is 1897 cm- 1 whereas, in the case of SDS it is observed to be 1984 cm- 1. The fluorescence decay curve of C2+ fits with multi exponential functions in the micellar system. The increase in lifetime of C2+ in SDS has been attributed to the increase in radiative rate due to the incorporation of C2+ at the micelle -water interface. The value of dynamic quenching constant determined is 16.9 M- 1. The location of the probe molecule in micellar systems has been justified by a variety of spectral parameters such as dielectric constant, ET (30), viscosity, EERS, average fluorescence decay time, radiative and non-radiative rate constants. All experimental results suggest that the C2+ molecule binds strongly with the SDS micelles and resides at micellar-water interface. The binding constant (Kb) calculated (3.85 × 105 M- 1) for C2+ in SDS revealed that the electrostatic forces mediate charge probe-micelle association.

Determination of physicochemical parameters of (bio)molecules and (bio)particles by capillary electromigration methods.

The review provides an overview of recent developments and applications of capillary electromigration (CE) methods for the determination of important physicochemical parameters of various (bio)molecules and (bio)particles. These parameters include actual and limiting (absolute) ionic mobilities, effective electrophoretic mobilities, effective charges, isoelectric points, electrokinetic potentials, hydrodynamic radii, diffusion coefficients, relative molecular masses, acidity (ionization) constants, binding constants and stoichiometry of (bio)molecular complexes, changes of Gibbs free energy, enthalpy and entropy and rate constants of chemical reactions and interactions, retention factors and partition and distribution coefficients. For the determination of these parameters, the following CE methods are employed: zone electrophoresis in a free solution or in sieving media, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography. In the individual sections, the procedures for the determination of the above parameters by the particular CE methods are described.

Contactless conductivity detector as a tool for improving universality and sensitivity of capillary electrophoresis-frontal analysis: Proof of concept.

Drug binding to plasma proteins influences processes such as liberation, adsorption, disposition, metabolism, and elimination of drugs, which are thus one of the key steps of a new drug development. As a result, the characterization of drug-protein interactions is an essential part of these time- and money-consuming processes. It is important to determine not only the binding strength and the stoichiometry of interaction, but also the binding site of a drug on a protein molecule, because two drugs with the same binding site can mutually affect free drug concentration. Capillary electrophoresis-frontal analysis with mobility shift affinity capillary electrophoresis is one of the most used affinity capillary electrophoresis methods for the characterization of these interactions. In this study, a well-known sensitivity problem of most capillary electrophoresis-frontal analyses using ultraviolet detection is solved by its combination with contactless conductivity detection, which provided sixfold lower limits of quantitation and detection. Binding parameters of the human serum albumin-salicylic acid model affinity pair were evaluated by this newly developed approach and by the classical approach with ultraviolet detection primarily used for their mutual comparison. The results of both approaches agreed well and are also in agreement with literature data obtained using different techniques.

Electroanalysis in Pharmacogenomic Studies: Mechanisms of Drug Interaction with DNA.

The review discusses electrochemical methods for analysis of drug interactions with DNA. The electroanalysis method is based on the registration of interaction-induced changes in the electrochemical oxidation potential of heterocyclic nitrogenous bases in the DNA molecule and in the maximum oxidation current amplitude. The mechanisms of DNA-drug interactions can be identified based on the shift in the electrooxidation potential of heterocyclic nitrogenous bases toward more negative (cathodic) or positive (anodic) values. Drug intercalation into DNA shifts the electrochemical oxidation potential to positive values, indicating thermodynamically unfavorable process that hinders oxidation of nitrogenous bases in DNA. The potential shift toward the negative values indicates electrostatic interactions, e.g., drug binding in the DNA minor groove, since this process does not interfere with the electrochemical oxidation of bases. The concentration-dependent decrease in the intensity of electrochemical oxidation of DNA bases allows to quantify the type of interaction and calculate the binding constants.

The avidin-theophylline complex: A structural and computational study.

The interaction between avidin and its counterpart biotin is one of central importance in biology and has been reproposed and studied at length. However, the binding pocket of avidin is prone to promiscuous binding, able to accommodate even non-biotinylated ligands. Comprehending the factors that distinguish the extremely strong interaction with biotin to other ligands is an important step to fully picture the thermodynamics of these low-affinity complexes. Here, we present the complex between chicken white egg avidin and theophylline (TEP), the xanthine derivative used in the therapy of asthma. In the crystal structure, TEP lies in the biotin-binding pocket with the same orientation and planarity of the aromatic ring of 8-oxodeoxyguanosine. Indeed, its affinity for avidin measured by isothermal titration calorimetry is in the same µM range as those obtained for the previously characterized nucleoside derivatives. By the use of molecular dynamic simulations, we have investigated the most important intermolecular interactions occurring in the avidin-TEP binding pocket and compared them with those obtained for the avidin 8-oxodeoxyguanosine and avidin-biotin complexes. These results testify the capability of avidin to complex purely aromatic molecules.

Elucidating the significance of molecular interaction between sulphur doped zinc oxide nanoparticles and serum albumin using multispectroscopic approach.

Ingenious nanomaterials with improved biocompatibility and multifunctional properties are gaining vital significance in biomedical applications, including advanced drug delivery and nanotheranostics. In a biological system, these nanoparticles interact with serum proteins forming a dynamic corona that affects their biological or toxicological properties producing undesirable effects. Thus, the current study focuses on the synthesis of sulphur-doped zinc oxide nanoparticles (ZnO/S NPs) and characterizing their mechanism of interaction with serum proteins using multispectroscopic approach. ZnO/S NPs were synthesized by employing a co-precipitation approach and characterized using various analytical techniques. The results of interaction studies demonstrated that ZnO/S NPs interact with serum albumins via the static quenching process. Analysis of thermodynamic parameters (ΔG, ΔH and ΔS) revealed that the binding process is spontaneous, exothermic and van der Waals force or hydrogen bonding plays a major role. The interaction of ZnO/S NPs with tyrosine residue in bovine serum albumin was established by synchronous fluorescence spectroscopy. In addition, the results of UV-visible, circular dichroism, Fourier transform infrared, Forster's resonance energy transfer theory and dynamic light scattering spectroscopic studies revealed that the ZnO/S NPs interact with albumin by inducing the conformational changes in secondary structure and reducing the α-helix content.

ß-Cyclodextrin and folic acid host-guest interaction binding parameters determined by Taylor dispersion analysis and affinity capillary electrophoresis.

The thermodynamic properties of molecular recognition in host-guest inclusion complexes can be studied by Taylor dispersion analysis (TDA). Host-guest inclusion complexes have modest size, and it is possible to get convergent results fast, achieving greater certainty for the obtained thermodynamic properties. Cyclodextrins (CDs) and their derivatives can be used as drug carriers that can boost stability, solubility, and bioavailability of physiologically active substances. A simple and effective approach for assessing the binding properties of CD complexes that are critical in the early stages of drug and formulation development is needed to fully understand the process of CD and guest molecules' complex formation. In this work, TDA was successfully used to rapidly determine interaction parameters, including binding constant and stoichiometry, between ß-CD and folic acid (FA) along with the diffusivities of the free FA and its complex with ß-CD. Additionally, the FA diffusion coefficient obtained by TDA was compared to the results previously obtained by nuclear magnetic resonance. Affinity capillary electrophoresis (ACE) was also used to compare the binding constants obtained by different methods. The results showed that the binding constants obtained by ACE were somewhat lower than those obtained by the two TDA procedures.

Homopurine guanine-rich sequences in complex with N-methyl mesoporphyrin IX form parallel G-quadruplex dimers and display a unique symmetry tetrad.

DNA can fold into G-quadruplexes (GQs), non-canonical secondary structures formed by π-π stacking of G-tetrads. GQs are important in many biological processes, which makes them promising therapeutic targets. We identified a 42-nucleotide long, purine-only G-rich sequence from human genome, which contains eight G-stretches connected by A and AAAA loops. We divided this sequence into five unique segments, four guanine stretches each, named GA1-5. In order to investigate the role of adenines in GQ structure formation, we performed biophysical and X-ray crystallographic studies of GA1-5 and their complexes with a highly selective GQ ligand, N-methyl mesoporphyrin IX (NMM). Our data indicate that all variants form parallel GQs whose stability depends on the number of flexible AAAA loops. GA1-3 bind NMM with 1:1 stoichiometry. The Ka for GA1 and GA3 is modest, ∼0.3 µM -1, and that for GA2 is significantly higher, ∼1.2 µM -1. NMM stabilizes GA1-3 by 14.6, 13.1, and 7.0 °C, respectively, at 2 equivalents. We determined X-ray crystal structures of GA1-NMM (1.98 Å resolution) and GA3-NMM (2.01 Å). The structures confirm the parallel topology of GQs with all adenines forming loops and display NMM binding at the 3' G-tetrad. Both complexes dimerize through the 5' interface. We observe two novel structural features: 1) a 'symmetry tetrad' at the dimer interface, which is formed by two guanines from each GQ monomer and 2) a NMM dimer in GA1-NMM. Our structural work confirms great flexibility of adenines as structural elements in GQ formation and contributes greatly to our understanding of the structural diversity of GQs and their modes of interaction with small molecule ligands.

The Study of the Effect of Dimethylsulfoxide (or Diethylsulfoxide) on Quinine Sulfate-DNA Binding by UV-Vis and Steady-State Fluorescence Spectroscopies.

The effect of dimethylsulfoxide (DMSO) and diethylsulfoxide (DESO) on binding between quinine sulfate (QS) and DNA was studied by virtue of UV-Vis absorption, steady-state fluorescence spectroscopies, and fluorescence polarization measurements. The binding constant was determined at three different temperatures and the values of standard Gibbs energy change, enthalpy and entropy of binding were determined. The mechanism of binding and the effect of sulfoxides on this process was revealed. The values of binding constant, fluorescence polarization and iodide quenching studies confirmed that the main binding mode in QS-DNA system is groove binding. Addition of sulfoxides does not change the binding mechanism. Moreover, with addition of sulfoxides binding constant increases due to the removal of water molecules from DNA grooves making them more available for QS molecules. To explain the effect of DMSO and DESO on QS-DNA binding the photophysical properties of QS in aqueous solutions of DMSO and DESO were also studied. On the basis of quantum yield of QS in water, DMSO and DESO the types of intermolecular interactions were discussed. The obtained results show that quantum yield of QS in sulfoxides is lower compared with that in water and aqueous solution of 0.1 M H2SO4. QS forms ground state complexes with both DMSO and DESO that are stronger fluorophores compared with free QS molecules.

Sensitivity enhancement of capillary electrophoresis-frontal analysis-based method for characterization of drug-protein interactions using on-line sample preconcentration.

Capillary electrophoresis-frontal analysis is one of the most frequently used approaches for the study of plasma protein-drug interactions as a substantial part of new drug development. However, the capillary electrophoresis-frontal analysis typically combined with ultraviolet-visible detection suffers from insufficient concentration sensitivity, particularly for substances with limited solubility and low molar absorption coefficient. The sensitivity problem has been solved in this work by its combination with an on-line sample preconcentration. According to the knowledge of the authors this combination has never been used to characterize plasma protein-drug binding. It resulted in a fully automated and versatile methodology for the characterization of binding interactions. Further, the validated method minimalizes the experimental errors due to a reduction in the manipulation of samples. Moreover, employing an on-line preconcentration strategy with capillary electrophoresis-frontal analysis using human serum albumin-salicylic acid as a model system improves the drug concentration sensitivity 17-fold compared to the conventional method. The value of binding constant (1.51 ± 0.63) · 104 L/mol obtained by this new capillary electrophoresis-frontal analysis modification is in agreement with the value (1.13 ± 0.28) ·104 L/mol estimated by a conventional variant of capillary electrophoresis-frontal analysis without the preconcentration step, as well as with literature data obtained using different techniques.

Interaction of Positively Charged Oligopeptides with Blood Plasma Proteins.

In this project, we combine two areas of research, experimental characterization and molecular docking studies of the interaction of positively charged oligopeptides with crucial blood plasma proteins. The investigated peptides are rich in NH2 groups of amino acid side chains from Dap, Orn, Lys, and Arg residues, which are relevant in protein interaction. The peptides are 9- and 11-mer with the following sequences: (Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt), (Lys-Dab-Ala-Gly-Orn-Pro-His-Lys-Arg), and (Lys-Dab-Dab-Gly-Orn-Pro-Phe(2-F)-Lys-Arg). The net charge of the compound strongly depends on the pH environment and it is an important aspect of protein binding. The studied oligopeptides exhibit therapeutic properties: anti-inflammatory activity and the capacity to diminish reactive oxygen species (ROS). Therefore, the mechanism of potential binding with blood plasma components is the next challenge. The binding interaction has been investigated under pseudo-physiological conditions with the main blood plasma proteins: albumin (BSA), α1-acid glycoprotein (AAG), and γ-globulin fraction (GGF). The biomolecular quenching constant (kq) and binding constant (Kb) were obtained by fluorescence spectroscopy at various temperatures. Simultaneously, the changes in the secondary structure of proteins were monitored by circular dichroism (CD) and infrared spectroscopy (IR) by quantity analysis. Moreover, molecular docking studies were conducted to estimate the binding affinity, the binding domain, and the chemical nature of these interactions. The results show that the investigated oligopeptides could be mainly transported by albumin, and the binding domain I is the most favored cavity. The BSA and GGF are able to form stable complexes with the studied compounds as opposed to AAG. The binding reactions are spontaneous processes. The highest binding constants were determined for Lys-Dab-Dab-Gly-Orn-Pro-His-Lys-Arg-Lys-Dbt peptide, in which the values of the binding constants Kb to BSA and GGF were 10.1 × 104 dm3mol-1 and 3.39 × 103 dm3mol-1, respectively. The positively charged surface of peptides participated in salt bridge interaction with proteins; however, hydrogen bonds were also formed. The secondary structure of BSA and GGF after contact with peptides was changed. A reduction in the α-helix structure was observed with an increase in the ß-sheet and ß-turn and random coil structures.

Extending the application potential of capillary electrophoresis/frontal analysis for drug-plasma protein studies by combining it with mass spectrometry detection.

CE/frontal analysis (CE/FA) is probably one of the most frequently used modes of CE for studying affinity interactions. It is typically performed with classic UV-Vis detection that suffers from low concentration sensitivity. To overcome this limitation, the applicability of CE/FA in combination with ESI-MS detection for the investigation of drug-HSA interactions was demonstrated. The developed new method combines the advantages of CE/FA, such as low sample consumption and no labeling or immobilization of interacting partners, with the benefits of MS detection, such as higher selectivity and sensitivity; moreover, it can be used for molecules lacking a fluorophore or chromophore. The binding parameters of tolbutamide (TL) and glimepiride (GLP), first- and second-generation antidiabetics that differ strongly in their solubility in aqueous solutions, were investigated by this CE/FA-MS method. This method, in contrast to the CE/FA method with the most commonly used UV-Vis detection, is more sensitive; an almost three times lower LOD was reached. The binding parameters of TL and GLP were investigated by this CE/FA-MS method and compared with the literature data. The binding constant value of TL obtained by UV-Vis detection was lower than the value obtained by the method hyphenated with MS detection, which is probably given by the influence of the ESI parameters on the stability of drug-HSA complex. In addition, the ratio of TL and HSA concentrations was divergent in both of the experimental approaches. Finally, it can be concluded that both detection methods have their strengths and weaknesses.

Interaction of Curcumin with Poly Lactic-Co-Glycolic Acid and Poly Diallyldimethylammonium Chloride By Fluorescence Spectroscopy.

Poly Lactic-Co-Glycolic Acid (PLGA) and Poly Diallyldimethylammonium Chloride (PDDA) are widely being used for drug delivery and curcumin is being studied as potential drug molecule for its anti-oxidant, anti-inflammatory and anti-cancer activities. The interaction between PLGA, PDDA and curcumin was investigated by fluorescence spectroscopy. The modified Stern-Volmer equation was used to estimate the value of the binding constant Ka and the van't Hoff equation was used to estimate the corresponding thermodynamic parameters (ΔHo, ΔSo, and ΔGo). The obtained results showed that the binding constant between PLGA and Curcumin is due to the formation of hydrogen bonds and van der Waals forces. However, PDDA interacts with curcumin through hydrophobic interactions. Moreover, zeta potential measurements were obtained for these polymers and the surface charge was compared in presence and absence of the negatively charged curcumin molecules. It was found that the results obtained by zeta potential measurements are in agreement with those obtained by fluorescence spectroscopy. It is also found that binding of curcumin with PDDA is further encouraged in the presence of PLGA.

Interactions between copper (II) and ß-amyloid peptide using capillary electrophoresis-ICP-MS: Kd measurements at the nanogram scale.

Although the interaction between the ß-amyloid peptide and copper (II) appears to play an important role in Alzheimer's disease, the affinity constant is still controversial and values are ranging from 107 to 1011 M-1. With the aim of clarifying this point, a complementary method, based on the capillary electrophoresis-ICP-MS hyphenation, was developed and competitive binding experiments were conducted in the presence of nitrilotriacetic acid. The effect of the capillary surface (neutral or positively charged) and nature of the buffer (Tris or Hepes) have been studied. Tris buffer was found to be inappropriate for such determination as it enhances the dissociation of copper (II) complexes, already occurring in the presence of an electric field in capillary electrophoresis. Using Hepes, a value of 1010 M-1 was found for the affinity of the small ß-amyloid peptide 1-16 for copper (II), which is in agreement with the values obtained for other proteins involved in neurodegenerative diseases. These constants were also determined in conditions closer to those of biological media (higher ionic strength, presence of carbonates).

Chiral analysis of ß-alanyl-d,l-tyrosine and its derivatives and estimation of binding constants of their complexes with 2-hydroxypropyl-ß-cyclodextrin by capillary electrophoresis.

Chiral CE methods were developed for the elucidation of l- or d-configuration of tyrosine residue in antimicrobial dipeptide ß-alanyl-tyrosine (ß-Ala-Tyr) isolated from the hemolymph of larvae of fleshfly Neobellieria bullata and for the evaluation of enantiopurity of its synthetic isomers (ß-Ala-d-Tyr and ß-Ala-l-Tyr), and enantiomers of their amidated and acetylated derivatives, ß-Ala-d,l-Tyr-NH2 and N-Ac-ß-Ala-d,l-Tyr, respectively. Baseline separations were achieved for all three pairs of enantiomers: (i) for ß-Ala-d,l-Tyr in acidic background electrolyte composed of 32/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 2.5, and 20 mg/mL 2-hydroxypropyl-ß-cyclodextrin as chiral selector; (ii) for ß-Ala-d,l-Tyr-NH2 enantiomers in acidic background electrolyte consisting of 48/50 mM tris(hydroxymethyl)aminomethane/H3 PO4 , pH 3.5, and 30 mg/mL 2-hydroxypropyl-ß-cyclodextrin; and (iii) for enantiomers of N-Ac-ß-Ala-d,l-Tyr in alkaline background electrolyte composed of 50/49 mM Na2 B4 O7 /NaOH, pH 10.5, and 60 mg/mL 2-hydroxypropyl-ß-cyclodextrin. From CE analyses of mixed samples of isolated ß-Ala-Tyr and synthetic standards ß-Ala-l-Tyr and ß-Ala-d-Tyr, it turned out that isolated ß-Ala-Tyr was pure l-enantiomer. In addition, the average apparent binding constants, Kb , and average actual ionic mobilities of the complexes of ß-Ala-d,l-Tyr and its above derivatives with 2-hydroxypropyl-ß-cyclodextrin were determined. These complexes were weak, with Kb values ranging from 11.2 to 79.1 L/mol. Their cationic mobilities were equal to (5.6-9.2) × 10-9 m2 /V/s, and anionic mobilities to (-1.3-1.6) × 10-9 m2 /V/s.

Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2.

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH-N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.

Interaction of Antibiotics and Humic Substances: Environmental Consequences and Remediation Prospects.

The occurrence and distribution of antibiotics in the environment has received increasing attention due to their potential adverse effects on human health and ecosystems. Humic substances (HS) influence the mobility, reactivity, and bioavailability of antibiotics in the environment significantly due to their interaction. As a result, HS can affect the dissemination of antibiotic-resistance genes, which is one of the main problems arising from contamination with antibiotics. The review provides quantitative data on the binding of HS with fluoroquinolones, macrolides, sulfonamides, and tetracyclines and reports the proposed mechanisms of their interaction. The main issues of the quantification of antibiotic-HS interaction are discussed, which are a development of standard approaches and the accumulation of a dataset using a standard methodology. This would allow the implementation of a meta-analysis of data to reveal the patterns of the binding of antibiotics to HS. Examples of successful development of humic-based sorbents for fluoroquinolone and tetracycline removal from environmental water systems or polluted wastewaters were given. Data on the various effects of HS on the dissemination of antibiotic-resistance genes (ARGs) were summarized. The detailed characterization of HS properties as a key point of assessing the environmental consequences of the formation of antibiotic-HS complexes, such as the dissemination of antibiotic resistance, was proposed.

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis.

The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5'-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.

Highly Selective Methodology for Entrapment and Subsequent Removal of Cobalt (II) Ions under Optimized Conditions by Micellar-Enhanced Ultrafiltration.

Micellar-enhanced ultrafiltration (MEUF), being a separation technique, was used to remove cobalt metal ion (Co2+) from their aqueous solutions in an application to reduce the toxicity level from industrial effluents using a micellar solution of anionic and cationic surfactants. The metal ions were first adsorbed by using anionic surfactants, i.e., sodium dodecyl sulfate (SDS) and sodium oleate (SO). The calculations for partition (Kx) and binding constants (Kb) and their respective free energy of partition and binding (ΔGp and ΔGb kJmol-1) helped significantly to find out the extent of binding or interaction of Co2+ with the surfactant and ΔGp and ΔGb were found to be -29.50 and -19.38 kJmol-1 for SDS and -23.95 and -12.67 kJmol-1 in the case of SO. MEUF work was also performed to find out the optimal conditions to remove metal pollutants from the aqueous system. For the said purpose, various factors and concentrations effect were studied, such as the concentration of the surfactant, concentration of the electrolyte (NaCl), transmembrane pressure, RPM, and pH. The efficiency of this process was checked by calculating various parameters, such as rejection percentage (R%) and permeate flux (J). A maximum rejection of 99.95% with SDS and 99.99% with SO was attained.