Specific Molecular Recognition Research Articles

Preparation of selective magnetic dispersive solid-phase sorbent and its application for recognition clenbuterol from bovine urine.

A new kind of selective magnetic dispersive solid-phase sorbent based on multiple Fe3O4 nanospheres as the core structure and molecular imprinted material as the shell structure was synthesized with tert-butylamine and 2-chloroaniline as the templates. The obtained multicore-shell-structured sorbent was spherical (diameter distribution 25-90μm) with porous morphologies, thus incorporating strong magnetic properties and specific molecular recognition coupled with rapid adsorption and dynamic equilibrium. The sorbent was applied for rapid and selective screening of clenbuterol (CLB) in bovine urine samples. Good linearity was obtained in the range 1.25-200ngmL(-1) with the average recovery at three spiked levels ranging from 91.4% to 105.3%. The proposed method significantly improved the purification and extraction efficiency of CLB in urine samples and eliminated the effect of template leakage during quantitative analysis.

Molecular Recognition of Parallel DNA Quadruplex d(TTAGGGT)4 by Mitoxantrone: Binding with 1:2 Stoichiometry Leading to Thermal Stabilization and Telomerase Inhibition

The interaction of the anthraquinone derivative mitoxantrone, a semisynthetic anti-cancer drug with two non-planar side chains, with heptamer G-quadruplex d(TTAGGGT)4 , which contains the human telomere DNA sequence, was evaluated by differential scanning calorimetry, fluorescence Job plotting, absorption, and NMR and CD spectroscopy. Binding led to thermal stabilization of DNA (ΔTm =13-20 °C). The spectra revealed that two mitoxantrone molecules bind externally at two sites of the DNA quadruplex as monomers, by partial insertion of the chromophore and side-chain interaction at the grooves. The inhibition of telomerase (IC50 =2 μM), as determined by a TRAP assay, can be attributed to thermal stabilization of the DNA quadruplex because of the interactions with mitoxantrone. The studies revealed highly specific molecular recognition between a ligand and a parallel-stranded G-quadruplex; this might serve as a platform for the rational design of new drugs.

Synergetic dual recognition and separation of the fungicide carbendazim by using magnetic nanoparticles carrying a molecularly imprinted polymer and immobilized β-cyclodextrin

The authors describe a nanomaterial for solid-phase extraction of carbendazim. Magnetic molecularly imprinted polymer nanoparticles (mag-MIP-NPs) were obtained by immobilizing the MIP and a thiolated β-cyclodextrin on the surface of magnetite (Fe3O4) nanoparticles coated with gold nanoparticles. Both the recognition sites of the MIP and the hydrophobic cavities in the β-cyclodextrin contribute to the specific molecular recognition and extraction of carbendazim. The mag-MIP-NPs have an apparent adsorption capacity of 190 mg⋅g‾1. Spiked vegetables were analyzed by using this material for extraction of carbendazim prior to its determination by ultra performance liquid chromatography (UHPLC). Recoveries range from 90.5 % to 109 %, and the detection limit is 3.0 pg⋅mL‾1.

Molecularly imprinted polymer for caffeic acid by precipitation polymerization and its application to extraction of caffeic acid and chlorogenic acid from Eucommia ulmodies leaves

Molecularly imprinted polymers (MIPs) for caffeic acid (CA) were prepared using 4-vinylpyridine and methacrylamide (MAM) as functional monomers, divinylbenzene as a crosslinker and acetonitrile–toluene (3:1, v/v) as a porogen by precipitation polymerization. The use of MAM as the co-monomer resulted in the formation of microsphere MIPs and non-imprinted polymers (NIPs) with ca. 3- and 5-μm particle diameters, respectively. Binding experiments and Scatchard analyses revealed that the binding capacity and affinity of the MIP to CA are higher than those of the NIP. The retention and molecular-recognition properties of the prepared MIPs were evaluated using water–acetonitrile and sodium phosphate buffer–acetonitrile as mobile phases in hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography, respectively. In HILIC mode, the MIP showed higher molecular-recognition ability for CA than in reversed-phase mode. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CA on the MIP in HILIC mode, while hydrogen bonding and hydrophobic interactions seem to work for the recognition of CA in reversed-phase mode. The MIP had a specific molecular-recognition ability for CA in HILIC mode, while other structurally related compounds, such as chlorogenic acid （CGA), gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP was successfully applied for extraction of CA and CGA in the leaves of Eucommia ulmodies in HILIC mode.

Antibody Recognition of Disordered Antigens

Disordered proteins are important antigens in a range of infectious diseases. Little is known, however, about the molecular details of recognition of disordered antigens by their cognate antibodies. Using a large dataset of protein antigens, we show that disordered epitopes are as likely to be recognized by antibodies as ordered epitopes. Moreover, the affinity with which antigens are recognized is, unexpectedly, only weakly dependent on the degree of disorder within the epitope. Structurally defined complexes of ordered and disordered protein antigens with their cognate antibodies reveal that disordered epitopes are smaller than their ordered counterparts, but are more efficient in their interactions with antibody. Our results demonstrate that disordered antigens are bona fide targets of antibody recognition, and that recognition of disordered epitopes is particularly sensitive to epitope variation, a finding with implications for the effects of disorder on the specificity of molecular recognition more generally.

Prediction of amino acid positions specific for functional groups in a protein family based on local sequence similarity.

The exchange of single amino acid residue in protein can substantially affect the specificity of molecular recognition. Many protein families can be divided into the groups based on specificity to recognized ligands. Prediction of group-discriminating residues within the certain family is extremely necessary for theoretical studies, enzyme engineering, drug design, and so on. The most existing methods use the multiple sequence alignment. They have the limitations in prediction accuracy due to the family sequence divergence and ligand-based grouping. We developed a new method SPrOS (Specificity Projection On Sequence) for estimating the specificity of residues to user-defined groups. SPrOS compares the sequence segments from the test protein and training proteins. Contrary to other segment-comparison approaches extracting the string motifs, SPrOS calculates the scores for single positions by the similarity of their surroundings. The method was evaluated on the simulated sequences and real protein families. The high-prediction accuracy was achieved for simulated sequences, in which SPrOS detected specific positions not predicted with the alignment-based method. For bacterial transcription factors (LacI/GalR) clearly divided into functional groups, the predicted specific residues corresponded to the published experimental data. In a more complicated case of protein kinases classified by inhibitor specificity, the positions predicted with high significance were located in ligand-binding areas. As the ligand specificity is not necessary coincided with phylogeny, evolutionary-coupled mutations could disturb the detection of ligand-specific residues. Excluding proximate homologs of the test protein kinase from the training set, we improved the prediction of the ligand-specific residues. The SPrOS is available at http://www.way2drug.com/spros/

Preparation and characterization of aptamer–polyelectrolyte films and microcapsules for biosensing and delivery applications

“Smart” materials are polymer systems that are able to change their physical or chemical properties in response to external stimuli in their environment. By adding a specific molecular recognition probe to a polymer, hybrid materials can be developed that retain the properties of the advanced polymer and gain the ability to respond to a specific molecular target. Aptamers are single-stranded oligonucleotides that are well-suited to serve as molecular recognition probes due to the specificity and affinity of their target recognition as well as their stability and ease of synthesis and labeling. In particular, their negatively charged backbone makes for their facile incorporation into polyelectrolyte-based materials. This article will provide a brief review of the currently reported biosensor and delivery platforms that have been reported employing aptamer–polyelectrolyte materials, as well as a detailed description of the methods used to synthesize and study films and microcapsules containing small-molecule aptamer probes.

Supramolecular Polymer Complexes Based on Specific Molecular Recognition: DNA and its Synthetic Mimics

Supramolecular Polymer Complexes Based on Specific Molecular Recognition: DNA and its Synthetic Mimics

Molecularly imprinted polymer for chlorogenic acid by modified precipitation polymerization and its application to extraction of chlorogenic acid from Eucommia ulmodies leaves

Molecularly imprinted polymers (MIPs) for chlorogenic acid (CGA) were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer, divinylbenzene as a crosslinker and methanol or dimethylsulfoxide as a co-solvent. The prepared MIPs were microspheres with a narrow particle size distribution. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high and low affinity sites, were formed on the MIP. The retention and molecular-recognition properties of the prepared MIP were evaluated using a mixture of water and acetonitrile as a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the retention factor of CGA was increased on the MIP. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CGA on the MIP. The MIP had a specific molecular-recognition ability for CGA, while other related compounds, such as caffeic acid, gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP for CGA was successfully applied for extraction of CGA in the leaves of Eucommia ulmodies.

Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells

Epidermal growth factor receptor (EGFR) plays an important role in signaling pathway of the development of breast cancer cells. Since EGFR overexpresses in most breast cancer cells, it is regarded as a biomarker molecule of breast cancer cells. Here we demonstrated a new AFM technique—topography and recognition (TREC) imaging—to simultaneously obtain highly sensitive and specific molecular recognition images and high-resolution topographic images of EGFR on single breast cancer cells.

Potential transducers based man-tailored biomimetic sensors for selective recognition of dextromethorphan as an antitussive drug.

A biomimetic potentiometric sensor for specific recognition of dextromethorphan (DXM), a drug classified according to the Drug Enforcement Administration (DEA) as a "drug of concern", is designed and characterized. A molecularly imprinted polymer (MIP), with special molecular recognition properties of DXM, was prepared by thermal polymerization in which DXM acted as template molecule, methacrylic acid (MAA) and acrylonitrile (AN) acted as functional monomers in the presence of ethylene glycol dimethacrylate (EGDMA) as crosslinker. The sensors showed a high selectivity and a sensitive response to the template in aqueous system. Electrochemical evaluation of these sensors revealed near-Nernstian response with slopes of 49.6±0.5 and 53.4±0.5 mV decade(-1) with a detection limit of 1.9×10(-6), and 1.0×10(-6) mol L(-1) DXM with MIP/MAA and MIP/AN membrane based sensors, respectively. Significantly improved accuracy, precision, response time, stability, selectivity and sensitivity were offered by these simple and cost-effective potentiometric sensors compared with other standard techniques. The method has the requisite accuracy, sensitivity and precision to assay DXM in pharmaceutical products.

Cell-Free Synthesis Meets Antibody Production: A Review

Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG) molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv) and antigen binding fragments (Fab), have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

Layer-by-layer fabrication of restricted access media-molecularly imprinted magnetic microspheres for magnetic dispersion microextraction of bisphenol A from milk samples

A novel hydrophilic BPA-imprinted magnetic microsphere was prepared with specific molecular recognition (2.54), water compatibility and exclusion biomacromolecules.

Generation of Electrochemical Gradient from Peptide Self-Assembly

The nucleating core of the Amyloid β peptide associated with Alzheimer's disease is able to organize into a peptide bilayer maintaining dimensions similar to biological phospholipid membranes. However, the dynamics of the hydrogen-bonded peptides are certainly different from the flexible alkanes of the lipid membranes, and this crystallinity prompted us to consider their dynamics and functional potential. Here we will review these unique surfaces built on cross-β structures of amyloid, and extend that pattern to mixing of positively- and negatively-charged surfaces to give even higher order architectures with high electrochemical potential across the 4 nm membrane. Further, we have developed EFM analyses to map the charge distribution and explored novel energy or electron transduction reactions. Our results show that these peptide membrane scaffolds are dynamic, and their ability to self-organize offers a new opportunity for engineering specific molecular recognition elements into the peptide assemblies and well-ordered materials.

A double-enhanced strip biosensor for the rapid and ultrasensitive detection of protein biomarkers.

Combining the specific molecular recognition reaction between biotin and streptavidin with a dual-class signal amplification system, containing the unique optical properties of gold nanoparticles and the catalytic reaction of horseradish peroxidase, a double-enhanced strip biosensor was designed for the first time for a visual and rapid detection of protein biomarkers with good accuracy, especially for CEA with a detection limit of 2.9 fg mL(-1).

Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy.

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.

Advances in cucurbituril bonded stationary phases for chromatographic separation

Cucurbit [n] urils (CB [n]), a kind of host molecules of the fourth generation supramolecule followed crown ethers, cyclodextrins and calixarenes in supramolecular chemistry, are macrocyclic ligands and consist of several glycoluril units. Their special molecular recognition based on unique separation selectivity and stability have gained great interest in supramolecular chemistry and chromatography. Hereby, we give a review of research progresses of cucurbit [n] urils structural features, its homologues and derivatives and their applications in chromatographic stationary phase.

Conformational diversity of bacterial FabH: Implications for molecular recognition specificity

The molecular basis of variable substrate and inhibitor specificity of the highly conserved bacterial fatty acid synthase enzyme, FabH, across different bacterial species remains poorly understood. In the current work, we explored the conformational diversity of FabH enzymes to understand the determinants of diverse interaction specificity across Gram-positive and Gram-negative bacteria. Atomistic molecular dynamics simulations reveal that FabH from E. coli and E. faecalis exhibit distinct native state conformational ensembles and dynamic behaviors. Despite strikingly similar substrate binding pockets, hot spot assessment using computational solvent mapping identified quite different favorable binding interactions between the two homologs. Our data suggest that FabH utilizes protein dynamics and seemingly minor sequence and structural differences to modulate its molecular recognition and substrate specificity across bacterial species. These insights will potentially facilitate the rational design and development of antibacterial inhibitors against FabH enzymes.

Determination of Some Flavonoids by HPLC Using Quercetin-Molecularly Imprinted Polymers

Three different molecularly imprinted polymers (MIPs) for quercetin were prepared with various combinations of functional monomers and porogens. First, the sorption capacities of investigated MIPs using frontal analyses were determined in different solvents. In order to asses the specificity of molecular recognition except of quercetin there was also employed another structurally related compound: rutin. In all cases it was observed that synthesized MIPs displayed the highest sorption capacities for the template in MeOH/H2O and in ACN for rutin. In the next step, the most optimal MIP (with 4-vinylpyridine as the functional monomer and acetone as the porogen) was chosen astheMISPE-sorbent to selective extraction of quercetin from the hop cone extract. Prepared polymeric material allowed the purification of sample with satisfactory capacity, selectivity to the template molecule, and a high reproducibility of results. LODs (S/N = 5) were 0.3 µg/mL for quercetin and 0.25 µg/mL for rutin. LOQs (S/N = 10) were 0.6 µg/mL for quercetin and 0.5 µg/mL for rutin.

Structural basis of degenerate specificity of an anti-sugar antibody

Although remarkable specificity of the acquired immune system was clearly being demonstrated, degenerate specificity in immune recognition is often been observed. We had started working on degenerate specificity of antibodies using peptide and sugar as a model system. Both carbohydrate antigen (me-α-Man) and peptide (DVFYPYPYASGS) were established to be equivalent in polyclonal as well as in mAb (2D10) responses (1). Thermodynamic analysis of Ag-Ab interactions had suggested the role of conformational flexibility (2) while crystallographic analysis indicated the importance of plasticity in the interactions (3) of the antigen combining site in the manifestation of molecular mimicry. It has been pointed out that even if the potential for flexibility existed, it was not being utilized while recognizing both ligands. So, in order to address this conundrum we started looking for other sugars and peptides that can bind to the mAb 2D10 with comparable affinities. Crystallographic analysis of 2D10 binding to five different sugars (me-α-Glc, α-Lac, α1-3-Mannobiose, α1-6-Mannobiose, α1-3, α1-6-Mannotriose) has given insights underlying the basis of specificity in molecular recognition. Comparison of all the structures has demonstrated that the antigen combining site for sugars is constituted of CDR H3, L1 and L3 only. All five sugars have an overlapping primary binding site (equivalent to me-α-Man interacting region). This primary sugar binding site has been shown to accommodate similar as well as dissimilar sugars by utilizing plasticity in the interacting residues available in the antigen combining site. The reducing sugar of the similar disaccharides (α1-3-Mannobiose, α1-6-Mannobiose) have been adjusted in the same direction but with utilizing different sets of interacting residues of the antibody paratope. However, the reducing sugar of a dissimilar disaccharide (α-Lac) exploits different paratope space altogether. The trisaccharide (α1-3, α1-6-Mannotriose) was accommodated in the same site by utilizing the conformational flexibility in the paratope region (mainly in CDR L1). This study had demonstrated that an affinity matured antibody may utilize at least three different strategies in order to accommodate structurally similar/dissimilar sugars.