Selectively Bind Research Articles

Complexes of G-Quadruplex DNA with Drug Like Molecules

Structural information on the complexes of drug like molecules with quadruplex DNAs can aid the development of therapeutics and research tools that selectively target specific quadruplex DNAs. Screening can identify candidate molecules that require additional evaluation. An enhanced hydroxyl radical cleavage protocol is demonstrated that can efficiently provide structural information on the complexes of the candidate molecules with quadruplex DNA. NMR methods have been used to offer additional structural information about the complexes as well as validate the results of the hydroxyl radical approach. This multi-step protocol has been demonstrated on complexes of the quadruplex formed by the thrombin binding aptamer (TBA) and promoter region of c-kit. The hydroxyl radical results indicate that NSC 176319, Cain's quinolinium that was found by screening, exhibits selective binding to the two TT loops. The NMR results are consistent with selective disruption of the hydrogen bonding between T4 and T13 as well as unstacking of these residues from the bottom quartet. The preliminary result indicates that the NSC 176319 also binds to the loops of the quadruplex formed by the promoter region of c-kit. NSC 176319 is used as a hit and the structurally similar molecules are being screened to find other candidate molecules. Thus, the combination of screening, hydroxyl radical footprinting and NMR can find new molecules that selectively bind to quadruplex DNAs as well as provide structural information about their complexes.

High-yield seedless synthesis of triangular gold nanoplates through oxidative etching.

We demonstrate that monodispersed triangular gold nanoplates with high morphological yield (>90%) can be synthesized through a rapid one-pot seedless growth process. The edge length of triangular Au nanoplates can be readily tuned between 40 and 120 nm by varying the reaction parameters. Systematic studies reveal that distinct from previous hypothesis that the formation of nanoplates is mainly determined by the selective binding of iodide ions, our results show that iodide ions could have dual functions: it can selectively bind to the Au {111} facets and also selectively remove other less stable shape impurities through oxidative etching by forming tri-iodide ions (I(3)(-)), thus facilitating the formation of nuclei with dominant planar structure. This new synthetic route will not only help to better understand the growth mechanism of triangular gold nanoplates but also promote the research in anisotropic noble metal nanostructures.

Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3′-terminal domain

Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3′-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA–protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed.

A crown ether decorated dibenzocoronene tetracarboxdiimide chromophore: synthesis, sensing, and self-organization.

A macrocyclic dibenzocoronene tetracarboxdiimide containing two benzo-21-crown-7 groups has been synthesized. It shows liquid-crystalline behavior and selectively binds Pb(2+) or K(+) to form 1:2 complexes in solution. The complexation leads to a significant increase of fluorescence; the surface organization of discotic columnar structures, in the solid-state, can be controlled by selective ion binding.

A Ca2+ controlled thioether linked bichromophoric squaraine foldamer for “turn on” fluorescent sensing of oxalate

Several thioether linked bichromophoric squaraines 1 were prepared and characterized. Among these, the bichromophore 1c, containing three sulfur atoms, was found to selectively bind with Ca2+ and lead to a folded conformation. The selective binding of Ca2+ resulted in an absorption blue shift and fluorescence quenching due to the cation-induced folding of the bichromophore and the resultant exciton coupling between the squaraine chromophores. Job's plot confirmed a 1:1 binding between 1c and Ca2+. Anions such as oxalate can extract Ca2+ from squaraine foldamers via the formation of insoluble salts and restore the fluorescent signal. This sensing approach can be applied to the “turn on” fluorescent detection of oxalate with a low detection limit of 5.2nM (3σ/k).

Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin.

The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors (“synthetic lectins”) constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.

Selective Binding of Amino Acids on Europium‐Substituted Polyoxometalates and the Interaction‐Induced Luminescent Enhancement Effect

AbstractA europium‐substituted polyoxometalate (K13[Eu(SiW10MoO39)2]⋅28 H2O, EuSiWMo) can selectively bind to basic amino acids, namely, lysine, arginine, and histidine, and induce the emission enhancement of Eu3+ significantly. The mechanism is attributed to electrostatic interactions and hydrogen bonds between basic residues of the amino acids and negative charges of EuSiWMo based on the 1H NMR titration spectra of amino acids and time‐resolved fluorescence decay curves of EuSiWMo.

Biofunctional nanofibrous substrate comprising immobilized antibodies and selective binding of autologous growth factors.

The immobilization of biomolecules at the surface of different biomedical devices has attracted enormous interest in order to enhance their biological functionality at the cellular level. This work aims to develop a biofunctional polymeric substrate capable of selectively binding growth factors (GFs) of interest from a pool of proteins present in a biological fluid: platelet lysate (PL). To achieve this goal, the surface of electrospun PCL nanofibers needs to be activated and functionalized to be able to insert chemical groups for the immobilization of antibodies. After determining the maximum immobilization capacity of each antibody, TGF-β1 (12 μg mL(-1)), bFGF (8 μg mL(-1)), and VEGF (4 μg mL(-1)), the next step was to confirm their bioavailability using recombinant proteins. The binding efficiency of PL-derived GFs was of 84-87% for TGF-β1, 55-64% for bFGF, and 50-59% for VEGF. Cellular assays confirmed the biological activity of the bound VEGF (both recombinant and PL-derived). Multiple antibodies (i.e., bFGF and VEGF) were also immobilized over the same structure in a mixed or side-by-side fashion. Using both autologous biological fluids and cells, it is possible to use this platform to implement very effective and personalized therapies that can be tailored to specific medical conditions.

Finding and characterizing the complexes of drug like molecules with quadruplex DNA: combined use of an enhanced hydroxyl radical cleavage protocol and NMR.

Structural information on the complexes of drug like molecules with quadruplex DNAs can aid the development of therapeutics and research tools that selectively target specific quadruplex DNAs. Screening can identify candidate molecules that require additional evaluation. An enhanced hydroxyl radical cleavage protocol is demonstrated that can efficiently provide structural information on the complexes of the candidate molecules with quadruplex DNA. NMR methods have been used to offer additional structural information about the complexes as well as validate the results of the hydroxyl radical approach. This multi-step protocol has been demonstrated on complexes of the chair type quadruplex formed by the thrombin binding aptamer, d(GGTTGGTGTGGTTGG). The hydroxyl radical results indicate that NSC 176319, Cain’s quinolinium that was found by screening, exhibits selective binding to the two TT loops. The NMR results are consistent with selective disruption of the hydrogen bonding between T4 and T13 as well as unstacking of these residues from the bottom quartet. Thus, the combination of screening, hydroxyl radical footprinting and NMR can find new molecules that selectively bind to quadruplex DNAs as well as provide structural information about their complexes.

Design Rules for Selective Binding of Nuclear Localization Signals to Minor Site of Importin α

Selectivity is a critical issue in molecular recognition. However, design rules that underlie selectivity are often not well understood. Here, we studied five classical nuclear localization signals (NLSs) that contain the motif KRx(W/F/Y)xxAF and selectively bind to the minor site of importin α. The selectivity for the minor site is dissected by building structural models for the NLS-importin α complexes and analyzing the positive design and negative design in the NLSs. In our models, the KR residues of the motif occupy the P1’ and P2’ pockets of importin α, respectively, forming hydrogen-bonding and cation-π interactions. The aromatic residue at the P4’ position plays dual roles in the selectivity for the minor site: by forming π-stacking with W357 of importin α to reinforce the minor-site binding; and by clashing with the P5 pocket in the major binding site. The F residue at the P8’ position occupies a deep pocket, providing additional stabilization. The P7’ position sits on a saddle next to the P8’ pocket and hence requires a small residue; the A residue fulfills this requirement. The principal ideas behind these blind predictions turn out to be correct in an evaluation against subsequently available X-ray structures for the NLS-importin α complexes, but some details are incorrect. These results illustrate that the selectivity for the minor site can be achieved via a variety of design rules.

Molecular Imprinting for High-Added Value Metals: An Overview of Recent Environmental Applications

One of the most hot topics of recent research is the reuse of some compounds existing as pollutants in environment. These compounds (molecules, ions, complexes, etc.) are of high-added value and it will be ideal to selectively bind them with any environmental application and reuse them in their initial or modified form. The latter can be achieved using molecular imprinting. In the present review article, an overview of the recent attempts for the selective binding of some precious metals (i.e., gold, silver, and platinum) of high-added value is done using molecular imprinted polymers (MIPs) as materials. The simplicity of their use, their relatively low cost, and the broad range of possible guest molecules (small organic molecules, ions, metals, and also biological macromolecules) have since led to the important development of molecular imprinting.

Nanoparticulate molecularly imprinted poly(acrylic acid) as potential stationary phase for chiral separation of ofloxacin

A novel method for the synthesis of nano size molecularly imprinted poly(acrylic acid) (PAA) by reverse microemulsion technique has been reported. Molecularly-imprinted PAA were also synthesised by bulk polymerisation. Levofloxacin (LOfx) (S ofloxacin) was used as the template for imprinting. Scanning electron micrograph and dynamic light scattering profile revealed that emulsion polymerisation resulted in nano size molecularly-imprinted polymer (MIP) with narrow size distribution whereas bulk polymerisation resulted in irregular shaped micron size particles. Rebinding of LOfx was highest for nano MIPs as compared to micron MIPs and the non-imprinted polymers (NIP). Imprinting factor which indicates selective binding capacity was maximum for the nano system both in water as well as in acetonitrile. Circular dichroism spectroscopy showed that nano and micron size MIPs could selectively bind to chiral LOfx from its racemic mixture (R, S ofloxacin). However, selectivity of nano MIPs was superior to micro MIPs. TLC plate prepared using nano MIP as the stationary phase could also isolate LOfx from Ofx as two distinct spots on the TLC plate, well separated spots were not visible when microsize or non-imprinted polymers were used as the stationary phase.

Structural Basis for Arsenate-Phosphate Discrimination

In Nature arsenate can in principle replace phosphate in its role as a key biological molecule (e.g. as in ATP) but arsenate analogues are less stable with respect to hydrolysis and reduction. Yet bacterial adoption of arsenic has been described and although the precise nature of the arsenic species is undetermined, it is possible that arsenate is incorporated as direct substitution for phosphate in cellular macromolecules. Several phosphate-binding proteins are able to selectively bind phosphate over arsenate despite their chemical similarities. We take advantage of this chemical similarity to elucidate arsenate-phosphate discrimination and to probe our understanding of phosphate-binding mechanisms in general. Specifically we consider a highly flexible synthetic hexapeptide capable of irreversibly binding phosphate as well as a number of highly selective phosphate binding proteins, including those with the evolutionarily conserved sequence known as a P-loop. A major challenge in structural analysis is that automatic ligand search methods are deemed to fail in detecting phosphate-binding sites in small and disordered phosphate-binding peptides. We use the Generalized Amber Force Field (GAFF) and extend it to include arsenate. Our ability to explore and explain experimental results using a combination of DFT, MD, and MCMC techniques are demonstrated in their ability to distinguish between the chemically close phosphate and arsenate ligands. Using a number computational techniques, we explore the large number of potential interactions that may be exploited to explain and control the specificity and selectivity of binding.

Using Thiol–Gold Bond Formation To Bridge Surfaces with a Polymer Brush: SFA Experiments and MD Simulations

Complementary interactions between functionalized surfaces are challenging to quantify, as both kinetics and thermodynamics are important. Optimal reaction parameters are key to the efficient formation of hierarchical nanostructures. Here, we report both surface forces apparatus (SFA) studies and molecular dynamics (MD) simulations of the controlled binding of thiol functionalized polymer brushes to complementary gold surfaces. Polymer brush–brush, brush–mica, and brush–gold experiments were performed in toluene. These comparative studies reveal the subtle balance of nonspecific and specific contributions to the measured interactions. Importantly, the physical phenomena responsible for the measured force profile in the selective binding system, including the formation of thiol–gold bonds, the resulting adhesion between the surfaces, the work to separate the surfaces, and the anchor strength of the polymer brush can be extracted. Corroborating MD simulations demonstrate that hysteresis in the approach and separation of these selectively bound surfaces is not the result of kinetic effects, but rather is due to the polydispersity of the brush itself and the resulting thiolated chain end distribution. We relate our findings to observations made in the formation of hierarchical particle aggregates.

Development of oligopeptide-based novel biosensor by solid-phase peptide synthesis on microchip

A miniaturized solid-phase peptide synthesis (SPPS) system was developed aiming a novel concept of programmable biosensor that can flexibly change the target on-site and on-demand. Oligopeptides, utilized as probes for the biosensor, were synthesized by the on-chip SPPS on a single bead trapped at the center of a micro channel. After the completion of probe synthesis, a sample solution containing target was exposed to the beads. The selective bindings of targets and the synthesized oligopeptide probes were optically measured. As examples of sensing, YOYO1 label DNA binding with three probe sequences RHKS, EDED (hydrophilic), LAGV (hydrophobic), and also Ni binding with His-tag were investigated. Sequence-dependent different affinity and selectively bindings of these targets were confirmed. These results demonstrated the concept of a programmable biosensor.

Rice WRKY13 Regulates Cross Talk between Abiotic and Biotic Stress Signaling Pathways by Selective Binding to Different cis-Elements

Plants use a complex signal transduction network to regulate their adaptation to the ever-changing environment. Rice (Oryza sativa) WRKY13 plays a vital role in the cross talk between abiotic and biotic stress signaling pathways by suppressing abiotic stress resistance and activating disease resistance. However, it is not clear how WRKY13 directly regulates this cross talk. Here, we show that WRKY13 is a transcriptional repressor. During the rice responses to drought stress and bacterial infection, WRKY13 selectively bound to certain site- and sequence-specific cis-elements on the promoters of SNAC1 (for STRESS RESPONSIVE NO APICAL MERISTEM, ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR1/2, CUP-SHAPED COTYLEDON), the overexpression of which increases drought resistance, and WRKY45-1, the knockout of which increases both bacterial disease and drought resistance. WRKY13 also bound to two cis-elements of its native promoter to autoregulate the balance of its gene expression in different physiological activities. WRKY13 was induced in leaf vascular tissue, where bacteria proliferate, during infection, and in guard cells, where the transcriptional factor SNAC1 enhances drought resistance, during both bacterial infection and drought stress. These results suggest that WRKY13 regulates the antagonistic cross talk between drought and disease resistance pathways by directly suppressing SNAC1 and WRKY45-1 and autoregulating its own expression via site- and sequence-specific cis-elements on the promoters of these genes in vascular tissue where bacteria proliferate and guard cells where the transcriptional factor SNAC1 mediates drought resistance by promoting stomatal closure.

Co-regulated gene expression by oestrogen receptor α and liver receptor homolog-1 is a feature of the oestrogen response in breast cancer cells

Oestrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, regulates breast cancer cell proliferation and promotes motility and invasion. To determine the mechanisms of LRH-1 action in breast cancer, we performed gene expression microarray analysis following RNA interference for LRH-1. Interestingly, gene ontology (GO) category enrichment analysis of LRH-1–regulated genes identified oestrogen-responsive genes as the most highly enriched GO categories. Remarkably, chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1 showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα−regulated genes by LRH-1 through binding to oestrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 overexpression stimulated ERα recruitment, while LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at oestrogen response elements controls the expression of oestrogen-responsive genes.

Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus

Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1(gp-340), mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.

Porphyrin-based receptors for selective ion bindings

The functional porphyrin derivatives in biological systems provide various motivations for the design of biomimetic functional materials. Using the porphyrin moieties, we recently have designed several types of receptor molecules for the bindings of anionic and cationic species. By the combination of hydrogen-bonding motifs and porphyrins, selective bindings of anionic species have been achieved by means of hydrogen bonds and electrostatic interactions. In this study, we are going to briefly review the results of recent researches on porphyrin-based host system for selective ion bindings.

Nanoantibiotic Particles for Shape and Size Recognition of Pathogens

ABSTRACTWe have developed a novel class of colloidal particles capable of shape and size recognition as well as specific binding to the target cells. These colloid particles were fabricated using a nanoimprinting technology which yields inorganic imprints of the chosen target microorganisms. The products of the templating process are partially fragmented inorganic shells which can selectively bind to their biological counterparts, therefore impairing microbial cell growth, replication and infection. We have named this class of particles, which are capable of selectively recognizing bacterial shape and size, “nanoantibiotics”, which can be further functionalized to kill the target cells. The selective binding is driven by the increased area of contact upon recognition of the cell shape and size between the cells and their matching inorganic shell fragments. Here, we demonstrate the cell recognition and binding action of such particles using two different microbial test organisms.