Expanding the scope of MS binding assays to low-affinity markers as exemplified for mGAT1

Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC–MS a procedure to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation of bound and unbound marker—the most crucial step of the assay—could be effectively achieved by filtration in a 96-well-format. MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using 1 mol L⁻¹ NaCl in the incubation milieu reveals remarkable differences with respect to the marker’s affinity and kinetics and to the investigated test compound’s potency. Principle of MS binding assays After incubation of a target with a native marker, bound and unbound marker are separated by filtration. Subsequently, the bound native marker is liberated from the target and finally quantified by LC-MS-MS. Dedicated to Prof. Hans-Dietrich Stachel on the occasion of his 80th birthday     

‘Morphs’ (MRFs): metal-reversible folding domains for differential IgG binding

BACKGROUND: Selective recognition and binding of IgG molecules is the basis for a host of immunological and affinity purification techniques. Capture of an IgG in these procedures relies chiefly on its interaction with one of a variety of reengineered bacterial receptors which bind to the Fc region of IgG molecules with very high affinity. While this interaction is extremely efficient in trapping IgG molecules, the tight interaction between the binding partners often requires denaturing conditions for disruption of the complex, which can adversely affect the yield of purified IgG and also limit the lifetime of the receptor matrix. An effective receptor/IgG binding system which could be modulated by less extreme conditions is of considerable general interest. RESULTS: We describe the properties of a series of modified Fc receptor domains which are competent to bind IgG with high affinity but which can be reversibly unfolded upon addition of modest amounts of transition metal ions. Data are presented demonstrating loss of the secondary structural content of the domains as a function of increasing metal concentration, with a concomitant decrease in IgG binding affinity. Variants of the Fc receptor differing at a single amino acid position display increased sensitivity to metal-induced unfolding, while retaining comparable IgG binding ability in the absence of metal. CONCLUSIONS: The interaction of this series of Fc receptors with metal ions abolishes IgG binding, but removal of metal ions allows refolding of the domains with restoration of the IgG binding function. Examples of the utility and potential applications of these metal-modulated IgG binding domains are discussed.     

Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets

StreptoTag is a novel affinity chromatography-based method for the isolation of high- and low-affinity RNA binding proteins. Originally it was shown possible to isolate recombinant protein from yeast or bacterial extracts using small, specific, well-characterised RNA binding targets. Here we show that using an enhanced aptamer it is not only possible to efficiently immobilise large, highly structured RNA binding targets onto the streptomycin columns but also that the StreptoTag method can be used for the isolation and purification of endogenously expressed regulatory proteins, with relatively low abundance, from eukaryotic extracts. As an example for this we uncover the identity of a karyophilic cellular protein which specifically binds to an area within the large, highly folded structure that characterises the mRNA from the unique 3' region (U3) of the mouse mammary tumour virus (MMTV) long terminal repeat (LTR). Hence, this method is now suitable for the quick and efficient isolation and identification of novel RNA binding proteins such as regulatory factors.     

Utilization of nanoparticle labels for signal amplification in ultrasensitive electrochemical affinity biosensors: A review

Nanoparticles with desirable properties not exhibited by the bulk material can be readily synthesized because of rapid technological developments in the fields of materials science and nanotechnology. In particular their highly attractive electrochemical properties and electrocatalytic activity have facilitated achievement of the high level of signal amplification needed for the development of ultrasensitive electrochemical affinity biosensors for the detection of proteins and DNA. This review article explains the basic principles of nanoparticle based electrochemical biosensors, highlights the recent advances in the development of nanoparticle based signal amplification strategies, and provides a critical assessment of the likely drawbacks associated with each strategy. Finally, future perspectives for achieving advanced signal simplification in nanoparticles based biosensors are considered.     

Analysis of drug interactions with serum proteins and related binding agents by affinity capillary electrophoresis: A review

Biomolecules such as serum proteins can interact with drugs in the body and influence their pharmaceutical effects. Specific and precise methods that analyze these interactions are critical for drug development or monitoring and for diagnostic purposes. Affinity capillary electrophoresis (ACE) is one technique that can be used to examine the binding between drugs and serum proteins, or other agents found in serum or blood. This article will review the basic principles of ACE, along with related affinity-based capillary electrophoresis (CE) methods, and examine recent developments that have occurred in this field as related to the characterization of drug–protein interactions. An overview will be given of the various formats that can be used in ACE and CE for such work, including the relative advantages or weaknesses of each approach. Various applications of ACE and affinity-based CE methods for the analysis of drug interactions with serum proteins and other binding agents will also be presented. Applications of ACE and related techniques that will be discussed include drug interaction studies with serum agents, chiral drug separations employing serum proteins, and the use of CE in hybrid methods to characterize drug binding with serum proteins.     

Efficient approach to produce multi‐functional copolymers for effective DNA binding

Norbornene‐derived copolymer with side‐chain phosphonic acid and cationic motifs (NORP‐PHOS‐CAT copolymer) are synthesized using ring opening metathesis polymerization method. All the monomers and polymers are characterized very carefully using nuclear magnetic resonance, MASS, Fourier transform infrared spectroscopy, and gel permeation chromatography technique. Thermogravimetric analysis, Fourier transform infrared spectroscopy, and scanning electron microscope techniques are employed to confirm the anchoring of Fe₃O₄ particles to the NORP‐PHOS‐CAT copolymer. Formation of nano‐aggregates from NORP‐PHOS‐CAT copolymer is first observed in dynamic light scattering, and later, it is confirmed as nano‐spheres by scanning electron microscope and transmission electron microscopy studies. Zeta‐potential values of nano‐spheres suggest that the cationic motifs are on the surface. The encapsulation of anionic dye methyl orange to the nanocarrier is analyzed through UV–Vis spectroscopy in aqueous medium. DNA binding nature of NORP‐PHOS‐CAT‐Fe copolymer is confirmed through the circular dichroism measurement and UV–Vis spectroscopy. To the best of our knowledge, this is the first report where the norbornene‐derived copolymers that are elegantly synthesized with both magnetic as well as cationic nature to demonstrate the effective encapsulation of dye into the nano‐spheres followed by its release. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis,structural and spectral properties of Au complexes: luminescence properties and their non‐covalent DNA binding studies

Gold complexes of 1,3‐ bis‐pyridylimidazolium chloride ( L1 ), 1,3‐bis‐[2,6‐diisopropylphenyl]imidazolium chloride ( L2 ) and 1,3‐bis‐[benzyl]benzimidazolium chloride ( L3 ) were synthesized and characterized by analytical methods. For the complexes, electronic spectral results show that there is a marked difference in the band feature observed in the spectra, ascribed to the greater relativistic effect of gold. In fluorescence studies, the complexes develop emission bands in the visible region (400–600 nm) after excitation at around 350 nm. Au complex–DNA binding was studied, and it was observed that genomic DNA isolated from the U373‐GB cell line was fragmented and in some cases degraded by the Au complexes. Furthermore, the intensity of the DNA band increased when concentration of the metal complex was augmented. This study shows that the DNA cleavage is mediated by the Au complex. Copyright © 2013 John Wiley & Sons, Ltd.     

Elongated Thrombin Binding Aptamer: A G‐Quadruplex Cation‐Sensitive Conformational Switch

Aptamer‐based biosensors offer promising perspectives for high performance, specific detection of proteins. The thrombin binding aptamer (TBA) is a G‐quadruplex‐forming DNA sequence, which is frequently elongated at one end to increase its analytical performances in a biosensor configuration. Herein, we investigate how the elongation of TBA at its 5′ end affects its structure and stability. Circular dichroism spectroscopy shows that TBA folds in an antiparallel G‐quadruplex conformation with all studied cations (Ba²⁺, Ca²⁺, K⁺, Mg²⁺, Na⁺, NH₄⁺, Sr²⁺ and the [Ru(NH₃)₆]^2+/3+ redox marker) whereas other structures are adopted by the elongated aptamers in the presence of some of these cations. The stability of each structure is evaluated on the basis of UV spectroscopy melting curves. Thermal difference spectra confirm the quadruplex character of all conformations. The elongated sequences can adopt a parallel or an antiparallel structure, depending on the nature of the cation; this can potentially confer an ion‐sensitive switch behavior. This switch property is demonstrated with the frequently employed redox complex [Ru(NH₃)₆]³⁺, which induces the parallel conformation at very low concentrations (10 equiv per strand). The addition of large amounts of K⁺ reverts the conformation to the antiparallel form, and opens interesting perspectives for electrochemical biosensing or redox‐active responsive devices.     

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies,and the Encapsulation and Release of Dyes through DNA‐Mediated Disassembly

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di‐ and monoimide chromophores and the investigation of their photophysical, self‐assembly and DNA‐binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG?dC)–poly(dG?dC) and poly(dA?dT)–poly(dA?dT). The interaction of these conjugates with DNA was evaluated by photo‐ and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5–8×10⁴ M ^?1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM , which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.     

Capillary electrophoresis methods for microRNAs assays: A review

MicroRNAs (miRNAs) are short noncoding RNAs that conduct important roles in many cellular processes such as development, proliferation, differentiation, and apoptosis. In particular, circulating miRNAs have been proposed as biomarkers for cancer, diabetes, cardiovascular disease, and other illnesses. Therefore, determination of miRNA expression levels in various biofluids is important for the investigation of biological processes in health and disease and for discovering their potential as new biomarkers and drug targets. Capillary electrophoresis (CE) is emerging as a useful analytical tool for analyzing miRNA because of its simple sample preparation steps and efficient resolution of a diverse size range of compounds. In particular, CE with laser-induced fluorescence detection is a promising and relatively rapidly developing tool with the potential to provide high sensitivity and specificity in the analysis of miRNAs. This paper covers a short overview of the recent developments and applications of CE systems in miRNA studies in biological and biomedical areas.     

Tri‐ and diorganotin(IV) derivatives of non‐steroidal anti‐inflammatory drug sulindac: Characterization,electronic structures (DFT), DNA binding and plasmid cleavage studies

Tri‐ and diorganotin(IV) derivatives of non‐steroidal anti‐inflammatory drug sulindac ( Sul ), coordinated with carboxylate oxygen, namely C₂₃H₂₅FO₃SSn ( 1 ), C₃₈H₃₁FO₃SSn ( 2 ), C₃₂H₄₃FO₃SSn ( 3 ), C₅₂H₄₂F₂O₆S₂Sn ( 4 ), C₄₄H₄₄S₂Cl₂O₆F₂Sn₂ ( 5 ), C₄₈H₅₀F₂O₆S₂Sn ( 6 ) and C₅₆H₆₆F₂O₆S₂Sn ( 7 ), have been synthesized and characterized using analytical and spectroscopic (IR, ¹H NMR, ¹³C NMR, ¹¹⁹Sn NMR and ESI‐MS) techniques. Optimized geometry and electronic structures of the complexes obtained from density functional theory calculations indicate that complexes 1 , 2 , 3 and 7 are tetra‐coordinated with monodentate carboxylates, 4 and 6 are hexa‐coordinated with highly distorted octahedral geometry, whereas 5 is penta‐coordinated with distorted trigonal bipyramidal geometry. Probable mode of DNA binding with ligand ( Sul ) and complexes 1 – 7 has been revealed via various biophysical techniques (UV–visible spectroscopy, fluorometry and circular dichroism). Intrinsic binding constants (K _b) obtained from UV–visible spectroscopy for Sul and complexes 1 – 7 are 3.69 × 10⁴, and 7.3 × 10³, 1.14 × 10⁴, 1.47 × 10⁴, 1.55 × 10⁴, 1.49 × 10⁴, 2.02 × 10⁴, 1.17 × 10⁴ M⁻¹, respectively. The quenching constants (K _sv) using fluorometric titrations, calculated from competitive binding of ethidium bromide versus Sul /complexes with calf thymus DNA, also correspond to the above results. Circular dichroism spectral patterns of calf thymus DNA with Sul and complexes 1 – 7 have also been investigated. All the results reveal that the complexes bind with DNA through partial intercalative mode. pBr322 plasmid fragmentation has also been studied using gel electrophoresis, which shows the fragmentation of circular DNA by an increase in nicked form and also by the appearance of linear form with increasing concentration of drug or complexes.     

A Signal‐Passing DNA‐Strand‐Exchange Mechanism for Active Self‐Assembly of DNA Nanostructures

DNA nanostructured tiles play an active role in their own self‐assembly in the system described herein whereby they initiate a binding event that produces a cascading assembly process. We present DNA tiles that have a simple but powerful property: they respond to a binding event at one end of the tile by passing a signal across the tile to activate a binding site at the other end. This action allows sequential, virtually irreversible self‐assembly of tiles and enables local communication during the self‐assembly process. This localized signal‐passing mechanism provides a new element of control for autonomous self‐assembly of DNA nanostructures.     

Cobalt(II)–metformin complexes containing α‐diimine/α‐diamine as auxiliary ligand: DNA binding properties

The cobalt(II) complexes [Co(Cl)₂(met)(o‐phen)] ( 1 ), [Co(Cl)₂(en)(met)] ( 2 ) and [Co(Cl)₂(met)(opda)] ( 3 ) (met = metformin, o‐phen = ortho‐phenanthroline, en = ethylenediamine, opda = ortho‐phenylenediamine) were synthesized and characterized using liquid chromatography–mass spectrometry, elemental analysis, molar conductance measurements, thermal analysis, infrared spectroscopy, magnetic moment measurements, electronic spectroscopy and X‐ray diffraction. The metal centre was found to be in an octahedral geometry. UV–visible absorption, fluorescence and viscosity measurements were conducted to assess the interaction of the complexes with calf thymus DNA. The complexes showed absorption hyperchromism in UV–visible spectra with DNA. The binding constants from UV–visible absorption studies were 1.38 × 10⁵, 2.1 × 10⁵ and 3.1 × 10⁵ M^?1 for 1 , 2 and 3 , respectively, and Stern–Volmer quenching constants from fluorescence studies were 0.146, 0.176 and 0.475, respectively. Viscosity measurements revealed that the binding of the complexes with DNA could be surface binding, mainly due to groove binding. The activities of the complexes in DNA cleavage decrease in the order 3 > 2 > 1 . The complexes were docked into DNA topoisomerase II using Discovery Studio 2.1 software.     

Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: A review

Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted.     

Methyl Benzoate Gallium(III) corrole complexes: DNA‐binding,Photocleavage Activity,Cytotoxicity on Tumor Cells

Two new gallium corrole complexes, 10‐(4‐Methoxycarbonylphenyl) ‐5, 15‐bis(pentafluorophenyl)corrolatogallium(III)( 1 ‐Ga) and 5,15‐bis(4‐Methoxycarbonylphenyl)‐10‐(pentafluorophenyl)corrolatogallium(III)( 2 ‐Ga), were synthesized and characterized. The interaction of these gallium corrole complexes with CT‐DNA was studied by fluorescence methods, UV–visible, viscosity measurements, molecular docking as well as agarose gel electrophoresis. The results revealed that both 1 ‐Ga and 2 ‐Ga interact with DNA via major groove binding and could cleavage the supercoiled plasmid DNA efficiently under irradiation. The inhibitor and singlet oxygen test indicated that singlet oxygen was the reactive oxygen species involved in the photocleavage DNA initiated by 1 ‐Ga or 2 ‐Ga. Cell viability experiments indicated that 1 ‐Ga and 2 ‐Ga show high photocytotoxicity and low dark toxicity towards tested QGY‐7701 and MHCC‐H/L tumor cell lines. Fluorescence probe tests showed the absorbed 1 ‐Ga and 2 ‐Ga in tumor cells are mainly localized in mitochondria, and the mitochondria membrane potential disruption was observed after irradiation.     

A representation transfer learning approach for enhanced prediction of growth hormone binding proteins

Growth hormone binding proteins (GHBPs) are soluble proteins that play an important role in the modulation of signaling pathways pertaining to growth hormones. GHBPs are selective and bind non-covalently with growth hormones, but their functions are still not fully understood. Identification and characterization of GHBPs are the preliminary steps for understanding their roles in various cellular processes. As wet lab based experimental methods involve high cost and labor, computational methods can facilitate in narrowing down the search space of putative GHBPs. Performance of machine learning algorithms largely depends on the quality of features that it feeds on. Informative and non-redundant features generally result in enhanced performance and for this purpose feature selection algorithms are commonly used. In the present work, a novel representation transfer learning approach is presented for prediction of GHBPs. For their accurate prediction, deep autoencoder based features were extracted and subsequently SMO-PolyK classifier is trained. The prediction model is evaluated by both leave one out cross validation (LOOCV) and hold out independent testing set. On LOOCV, the prediction model achieved 89.8%% accuracy, with 89.4% sensitivity and 90.2% specificity and accuracy of 93.5%, sensitivity of 90.2% and specificity of 96.8% is attained on the hold out testing set. Further a comparison was made between the full set of sequence-based features, top performing sequence features extracted using feature selection algorithm, deep autoencoder based features and generalized low rank model based features on the prediction accuracy. Principal component analysis of the representative features along with t-sne visualization demonstrated the effectiveness of deep features in prediction of GHBPs. The present method is robust and accurate and may complement other wet lab based methods for identification of novel GHBPs.     

The formation of a metallosupramolecular porous helicate through salicylaldehydethiosemicarbazone: Synthesis,Characterization, Cytotoxic activity,DNA binding and DFT calculations

The reaction of salicylaldehyde‐S‐methylisothiosemicarbazone in the presence of ethylenediamine base and iron (III)chloride generated unforeseen homotopic dinuclear triple‐stranded iron (III)helicate. The synthesized helicate was characterized by elemental analysis, IR, UV–Vis spectroscopy, magnetic moment measurement, and evaluated cytotoxic activities against K562, HL‐60 and THP‐1 leukemia cells. In addition, solid‐state structure has been determined by single‐crystal X‐ray diffraction technique. In the complex, three dinucleating O, N, N, O donor ligands provide three diazine (═N─N═) bridges between the metal ions and facial O₃N₃ coordination spheres around them. The ligands are folded about the N ─ N single bond and coordinated to the two metal ions in a helical fashion to form the triple helical structure. In the crystal lattice, chains of centrosymmetric rings, which are connected to one another via π─π stacking interactions, are generated by C─H···O intermolecular interactions. The results are also confirmed by the density functional theory (DFT) calculations. The results obtained from the cytotoxicity test showed to be effective in low concentrations on the leukemia cells. An intercalative binding mode of helicate‐DNA complex was confirmed with the high intrinsic binding constant (K_b = 8×10⁶ M^?1) and competitive displacement assay of Ethidium bromide with high Ksv value.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.     

A Label‐Free Electrochemical Aptasensor for Thrombin Using a Single‐Wall Carbon Nanotube (SWCNT) Casted Glassy Carbon Electrode (GCE)

An electrochemical aptasensor with a thrombin binding aptamer (TBA) was developed using a single‐wall carbon nanotube (SWCNT) casted GCE. The TBA was immobilized on SWCNTs through π‐stacking without any special modification, resulting in helical wrapping to the surface. In the presence of thrombin, the TBA binds with thrombin and the TBA concentration on the SWCNT surface decreases. The remaining amount of TBA can be analyzed by an electrochemical method without any label, because the guanine bases of the nucleic acid are measurable by electrochemical methods. The electrochemical oxidation of guanine nucleotides was enhanced by electrocatalytic mediation using Ru(bpy)₃²⁺ for higher sensitivity and reduction of the overpotential for electrochemical detection.