Site-specific Reaction Research Articles

Extracellular domain of human calcium sensing receptor immobilized to silica beads as biomaterial: A rapid chromatographic method for recognizing ligands from complex matrix ‘Shuangdan’

Human calcium-sensing receptor (CaSR), a member of the G-protein-coupled receptor superfamily (GPCR), has been a therapeutic target for developing new drugs against calciotropic disorders and non-calciotropic diseases. The highly efficient methodologies for pursuing novel ligands/drugs remained a challenge due to the redundant purification processes of membrane protein in some widely-used methods including NMR, X-ray crystallography, Fluorescence Titration Spectroscopy, and Circular Dichroism. Herein, extracellular domain (ECD) of CaSR as its functional fragment was used to develop a rapid chromatographic method, which involved the synthesis of stationary phase material based on the site-specific covalent reaction of Halogenated alkane dehalogenase (Halo)-tagged ECD of CaSR in cell lysate with 6-chlorocaproic acid modified silica beads, the use of the immobilized CaSR column for revealing the interaction of three known agonists with CaSR and further screening ligands from complex matrix like Chinese herb medicine ‘Shuangdan’. The immobilized CaSR column was prepared rapidly without the protein purification and retained a good stability and specificity for at least 35 days. It was revealed that one type of binding sites occurred on CaSR with the binding affinity of neomycin > gentamicin-C / kanamycin, presumably which related to the number of structural amino groups attached. This method allowed for recognizing specifically novel ligands from ’Shuangdan’, demonstrating one type of binding sites on CaSR with the binding affinity of gallic acid > caffeic acid > paeonol. These results indicated that, the immobilization of a representative extracellular domain of CaSR to silica beads as biomaterial is feasible to develop a new rapid method, which can be successfully applied in screening novel ligands efficiently from complex matrices.

Cationic peptide carriers enable long-term delivery of insulin-like growth factor-1 to suppress osteoarthritis-induced matrix degradation

BackgroundInsulin-like growth factor-1 (IGF-1) has the potential to be used for osteoarthritis (OA) treatment but has not been evaluated in clinics yet owing to toxicity concerns. It suffers from short intra-joint residence time and a lack of cartilage targeting following its intra-articular administration. Here, we synthesize an electrically charged cationic formulation of IGF-1 by using a short-length arginine-rich, hydrophilic cationic peptide carrier (CPC) with a net charge of +14, designed for rapid and high uptake and retention in both healthy and arthritic cartilage.MethodsIGF-1 was conjugated to CPC by using a site-specific sulfhydryl reaction via a bifunctional linker. Intra-cartilage depth of penetration and retention of CPC-IGF-1 was compared with the unmodified IGF-1. The therapeutic effectiveness of a single dose of CPC-IGF-1 was compared with free IGF-1 in an IL-1α-challenged cartilage explant culture post-traumatic OA model.ResultsCPC-IGF-1 rapidly penetrated through the full thickness of cartilage creating a drug depot owing to electrostatic interactions with negatively charged aggrecan-glycosaminoglycans (GAGs). CPC-IGF-1 remained bound within the tissue while unmodified IGF-1 cleared out. Treatment with a single dose of CPC-IGF-1 effectively suppressed IL-1α-induced GAG loss and nitrite release and rescued cell metabolism and viability throughout the 16-day culture period, while free IGF at the equivalent dose was not effective.ConclusionsCPC-mediated depot delivery of IGF-1 protected cartilage by suppressing cytokine-induced catabolism with only a single dose. CPC is a versatile cationic motif that can be used for intra-cartilage delivery of other similar-sized drugs.

Finding efficient catalyst designs: A high-precision method to reveal active sites

Designing efficient electrocatalytic structures requires reliable guidelines. For this purpose, experimental approaches for the characterization of model electrodes in operando conditions are valuable. Reporting in Joule , Agnoli et al. showcase the determination of site-specific reaction onset potentials and Tafel slopes by using electrochemical scanning tunneling microscopy. Designing efficient electrocatalytic structures requires reliable guidelines. For this purpose, experimental approaches for the characterization of model electrodes in operando conditions are valuable. Reporting in Joule , Agnoli et al. showcase the determination of site-specific reaction onset potentials and Tafel slopes by using electrochemical scanning tunneling microscopy.

Structure and mechanism of a methyltransferase ribozyme

Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

Water-Soluble [Tc(N)(PNP)] Moiety for Room-Temperature 99mTc Labeling of Sensitive Target Vectors.

The incorporation of bioactive molecules into a water-soluble [99mTc][Tc(N)(PNP)]-based mixed compound is described. The method, which exploits the chemical properties of the new [99mTc][Tc(N)(PNP3OH)]2+ synthon [PNP3OH = N,N-bis(di-hydroxymethylenphosphinoethyl)methoxyethylamine], was successfully applied to the labeling of small, medium (cysteine-functionalized biotin and c-RGDfK pentapeptide), and large molecules. Apomyoglobin was chosen as a model protein and derivatized via site-specific enzymatic reaction catalyzed by transglutaminase (TGase) with the H-Cys-Gly-Lys-Gly-OH tetrapeptide for the insertion in the protein sequence of a reactive N-terminal Cys for 99mTc chelation. Radiosyntheses were performed under physiological conditions at room temperature within 30 min. They were reproducible, highly specific, and quantitative. Heteroleptic complexes are hydrophilic and stable. Biodistributions of the selected compounds show favorable pharmacokinetics within 60 min post-injection and predominant elimination through the renal-urinary pathway. In a wider perspective, these data suggest a role of the [99mTc][Tc(N)(PNP)] technology in the labeling of temperature-sensitive biomolecules, especially targeting proteins for SPECT imaging.

Site-Specific Photochemical Reaction for Improved C=C Location Analysis of Unsaturated Lipids by Ultraviolet Photodissociation.

Unraveling the complexity of the lipidome requires the development of novel approaches to facilitate structural identification and characterization of lipid species with isomer-level discrimination. Ultraviolet photodissociation tandem mass spectrometry (UVPD MS/MS) is a promising tool for structure determination of lipids. The sensitivity of UVPD for lipid analysis however is limited mainly due to weak absorption of UV photons by a C=C. Herein, a C=C site-specific derivatization, the Paternò-Büchi (PB) reaction, was used to incorporate a chromophore to the C=C moiety in fatty acyls, leading to significantly improved UVPD efficiency and sensitivity for pinpointing C=C locations. The wavelength-dependent photodissociation of the PB products demonstrated 4-CF3-benzophenone as the best reagent for UVPD in terms of the efficiency of generating C=C diagnostic fragments and simplicity for C=C location assignments. We demonstrated the effectiveness of this approach for the shotgun profiling of C=C location isomers in different lipid classes from complex lipid extracts, highlighting its potential to advancing the identification of the C=C bond locations in unsaturated lipids.

An RNA-cleaving threose nucleic acid enzyme capable of single point mutation discrimination.

Threose nucleic acid has been considered a potential evolutionary progenitor of RNA because of its chemical simplicity, base pairing properties and capacity for higher-order functions such as folding and specific ligand binding. Here we report the in vitro selection of RNA-cleaving threose nucleic acid enzymes. One such enzyme, Tz1, catalyses a site-specific RNA-cleavage reaction with an observed pseudo first-order rate constant (kobs) of 0.016 min-1. The catalytic activity of Tz1 is maximal at 8 mM Mg2+ and remains relatively constant from pH 5.3 to 9.0. Tz1 preferentially cleaves a mutant epidermal growth factor receptor RNA substrate with a single point substitution, while leaving the wild-type intact. We demonstrate that Tz1 mediates selective gene silencing of the mutant epidermal growth factor receptor in eukaryotic cells. The identification of catalytic threose nucleic acids provides further experimental support for threose nucleic acid as an ancestral genetic and functional material. The demonstration of Tz1 mediating selective knockdown of intracellular RNA suggests that functional threose nucleic acids could be developed for future biomedical applications.

Precise quantification of N1-Methyladenosine with a site-specific RNase H cleavage-assisted isothermal amplification strategy

Specific and accurate determination of N1-Methyladenosine (m1A) is of critical significance to better understand its biological functions. Precise quantification of low level of RNA modification against a high background of homologous normal RNA still remains a big challenge. To solve this issue, a facile site-specific cleavage-assisted isothermal exponential amplification reaction (SSC-EXPAR) is skillfully designed for direct m1A assay. By exploiting the highly selective RNA cleavage, the normal RNA can be disconnected to avoid the false-positive interference, while the intact m1A can mediate and trigger three-way junction structure-based EXPAR (3WJ-EXPAR). The elegant design of m1A-aroused positive signal can guarantee the accurate and reliable detection of low-level m1A. As low as 1 fM of m1A-containing RNA can be accurately determined with a specificity allowing the discrimination of 0.5% m1A against unmodified adenosine (A) at single-base resolution. In virtue of the outstanding sensitivity and specificity, the SSC-EXPAR approach holds great potential in further understanding of m1A-associated biofunctions and the clinical value in diagnosis and therapeutic treatment of human cancers.

Predicting Rate Constants of Hydroxyl Radical Reactions with Alkanes Using Machine Learning.

The hydrogen abstraction reactions of the hydroxyl radical with alkanes play an important role in combustion chemistry and atmospheric chemistry. However, site-specific reaction constants are difficult to obtain experimentally and theoretically. Recently, machine learning has proved its ability to predict chemical properties. In this work, a machine learning approach is developed to predict the temperature-dependent site-specific rate constants of the title reactions. Multilayered neural network (NN) models are developed by training the site-specific rate constants of 11 reactions, and several schemes are designed to improve the prediction accuracy. The results show that the proposed NN models are robust in predicting the site-specific and overall rate constants.

Site-Specific Scissors Based on Myeloperoxidase for Phosphorothioate DNA.

The tool box of site-specific cleavage for nucleic acid has been an increasingly attractive subject. Especially, the recent emergence of the orthogonally activatable DNA device is closely related to the site-specific scission. However, most of these cleavage strategies are based on exogenous assistance, such as laser irradiation. Endogenous strategies are highly desirable for the orthogonally regulatable DNA machine to explore the crucial intracellular biological process and cell signal network. Here, we found that the accurate site-specific cleavage reaction of phosphorothioate (PT) modified DNA by using myeloperoxidase (MPO). A scissors-like mechanism by which MPO breaks PT modification through chloride oxidation has been revealed. Furthermore, we have successfully applied the scissors to activate PT-modified hairpin-DNA machines to produce horseradish peroxidase (HRP)-mimicking DNAzyme or initiate hybridization chain reaction (HCR) amplification. Since MPO plays an important role in the pathway related to oxidative stress in cells, through the HCR amplification activated by this tool box, the oxidative stress in living cells has been robustly imaged. This work proposes an accurate and endogenous site-specific cleavage tool for the research of biostimuli and the construction of DNA molecular devices.

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli.

We describe a new set of tools for inserting DNA into the bacterial chromosome. The system uses site-specific recombination reactions carried out by bacteriophage integrases to integrate plasmids at up to eight phage attachment sites in E. coli MG1655. The introduction of mutant loxP sites in the integrating plasmids allows repeated removal of antibiotic resistance genes and other plasmid sequences without danger of inducing chromosomal rearrangements. The protocol for Cre-mediated antibiotic resistance gene removal is greatly simplified by introducing the Cre plasmid by phage infection. Finally, we have also developed a set of four independently inducible expression modules with tight control and high dynamic range which can be inserted at specific chromosomal locations.

Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

DNAzymes as key components of biosensing systems for the detection of biological targets

DNAzymes are synthetic functional nucleic acids that have found widespread use in biosensing applications both for molecular recognition and signal generation. Two classes of DNAzymes have proved particularly effective for use in proof-of-concept biosensing systems, namely RNA-cleaving DNAzymes (RCDs) and peroxidase mimicking DNAzymes (PMDs). RCDs catalyze the site-specific cleavage reaction of an RNA dinucleotide junction, generating two cleavage fragments. PMDs are capable of catalyzing peroxidation reactions of chromophores, thereby generating a measurable signal. Herein, we review the use of these DNAzymes in biomedical assays and diagnostics, and show that this emerging field should have great promise for biosensor development over the next few decades.

Site-specific soil reaction model for monopiles in soft clay based on laboratory element stress-strain curves

Large-diameter monopiles are the predominant foundation solution for supporting offshore wind turbines. They are conventionally designed using soil reaction curves developed for long slender piles used for supporting offshore oil and gas platforms (e.g., the API p-y model). However, due to the difference in the length/diameter ratio and the resulting soil mechanisms, the use of p-y curves alone can lead to significant under-prediction of the lateral stiffness and capacity of monopiles. To overcome the shortcoming, the authors have previously proposed a conceptual two-spring framework, i.e., the so-called ‘p-y + MR-θR’ model, to capture the monopile response in soft clay under lateral loading. The framework uses distributed p-y springs to consider the lateral soil resistance along the pile above the rotation point (RP) and a single moment-rotation (MR-θR) spring attached at the RP to capture the entire soil resistance below the RP, i.e., the distributed resistance along the pile, base shear and base moment at the pile tip. The proposed p-y and MR-θR springs were curve-fitted to the results of 3D numerical analyses. However, as the stress-strain response and the shear strength profile inevitably influence the p-y and MR-θR springs, the applicability of the empirical formulations to soil conditions other than those examined is uncertain. This study proposes an enhancement to the ‘p-y + MR-θR’ framework, in which the p-y and MR-θR springs are not tied to a specific soil and strength profile but fundamentally linked to the properties that can be measured directly in the site investigation and laboratory. This extension is achieved through analytical analyses and an extensive parametric numerical study. The predictive capabilities of the model are demonstrated by back-analyses of finite element analyses and centrifuge model tests. The proposed model provides practising engineers with a simple yet powerful approach to use site-specific soil reaction curves in the design of monopiles embedded in soft clay.

FTIR Characterization of the Development of Antimicrobial Catheter Coatings Loaded with Fluoroquinolones

The purpose of this paper was to present the development of antimicrobial coatings for different urinary catheters. Antimicrobial catheter coatings were prepared by immobilizing fluoroquinolones either with the use of linkers (covalent binding) or by activating the polymer matrix with iodine/bromine (noncovalent binding). The possibility of the deposition of antimicrobial agent(s) following bromine activation on latex, polyurethane, and silicone was evaluated. Fourier transform infrared spectroscopy (FTIR), used to monitor the changes in the catheter’s molecular structure occurring over the course of its multi-stage modification, confirmed the presence of fluoroquinolones in the catheter matrix as well as site-specific reactions. The amounts of drugs embedded in the catheter matrix were determined by the HPLC method. Stability of the drug binding was checked by examining the drug release. The new antimicrobial coatings obtained with the participation of fluoroquinolone antibiotics have the potential to protect the patient against infections during catheterization.

Co-transcriptional Analysis of Self-Cleaving Ribozymes and Their Ligand Dependence.

Self-cleaving ribozymes are RNA molecules that catalyze a site-specific self-scission reaction. Analysis of self-cleavage is a crucial aspect of the biochemical study and understanding of these molecules. Here we describe a co-transcriptional assay that allows the analysis of self-cleaving ribozymes in different reaction conditions and in the presence of desired ligands and/or cofactors. Utilizing a standard T7 RNA polymerase in vitro transcription system under limiting Mg2+ concentration, followed by a 25-fold dilution of the reaction in desired conditions of self-cleavage (buffer, ions, ligands, pH, temperature, etc.) to halt the synthesis of new RNA molecules, allows the study of self-scission of these molecules without the need for purification or additional preparation steps, such as refolding procedures. Furthermore, because the transcripts are not denatured, this assay likely yields RNAs in conformations relevant to co-transcriptionally folded species in vivo.

IHF stabilizes pathogenicity island I of uropathogenic Escherichia coli strain 536 by attenuating integrase I promoter activity

Pathogenicity islands (PAIs) represent horizontally acquired chromosomal regions and encode their cognate integrase, which mediates chromosomal integration and excision of the island. These site-specific recombination reactions have to be tightly controlled to maintain genomic stability, and their directionality depends on accessory proteins. The integration host factor (IHF) and the factor for inversion stimulation (Fis) are often involved in recombinogenic complex formation and controlling the directionality of the recombination reaction. We investigated the role of the accessory host factors IHF and Fis in controlling the stability of six PAIs in uropathogenic Escherichia coli strain 536. By comparing the loss of individual PAIs in the presence or absence of IHF or Fis, we showed that IHF specifically stabilized PAI I536 and that in particular the IHFB subunit seems to be important for this function. We employed complex genetic studies to address the role of IHF in PAI I536-encoded integrase (IntI) expression. Based on different YFP-reporter constructs and electrophoretic mobility shift assays we demonstrated that IntI acts a strong repressor of its own synthesis, and that IHF binding to the intI promoter region reduces the probability of intI promoter activation. Our results extend the current knowledge of the role of IHF in controlling directionality of site specific recombination reactions and thus PAI stability.

Site-selective, reversible, pH-induced N-terminal maleylation and its application for proteomics research.

Compared with traditional labelling reagents used in proteomics, maleic anhydride is milder and can be easily removed under certain conditions, thus simplifying chemical derivatization. The proposed strategy combined a site-specific chemical labelling reaction with mass spectrometry. Site-selective, reversible N-terminal maleylation was controlled by pH. Selective maleyl N-terminal labelling was achieved with high efficiency under the optimized reaction conditions. The demaleylation conditions were also optimized. The sequence coverage of histone H4 increased from 77% to 95% after the maleyl labels were removed, and the number of maleylated peptides was five times that of the unlabelled peptides. We further verified the reversible and selective N-terminal labelling properties of maleic anhydride through propionylation labelling at the peptide/protein level. A new method for site-selective maleylation of the N-terminal amino groups of a peptide was explored. Through the optimization experiment, good efficiency was achieved for this labelling reaction. The reversibility of maleylation labelling was also explored and applied for the identification of post-translational modifications of histones. Thus, site-selective, reversible, pH-induced N-terminal labelling using maleic anhydride has greater potential for application in proteomics than any other labelling methods.

Uncertainty and sensitivity analysis of the chemistry of cesium sorption in deep geological repositories

Sorption is the most important process responsible for the retention of radionuclides or heavy metals in repositories. A major task in understanding sorption is to account for uncertainties that affect the long-term repository performance, and eventually, study their effect on the migration of radionuclides. This paper presents a novel comprehensive uncertainty and sensitivity analysis study of a detailed sorption chemistry model used in Cesium reactive transport modeling, based on the Morris and Sobol method. The analysis addresses the effects on the transport of Cesium through clay, exemplified for Cesium migration in the vicinity of a deep geological repository for nuclear waste, due to uncertainties in 1) site-specific detailed equilibrium sorption reactions constants and 2) clay pore water cations’ concentration, i.e. the pore water composition. The results show that the equilibrium sorption reaction constants on Type2 and FES sites in addition to the concentrations of the respective cations involved in these surface reactions are the most important uncertain parameters affecting the transport of Cesium. Furthermore, a classification tree is constructed to identify the combination of parameters values leading to the maximum Cesium concentrations at a specified location. Finally, a new Cesium isotherm and Kduncertainty range are constructed numerically based on our deterministic complex sorption chemistry calculations using the MCOTAC reactive transport code. These results can be used in large-scale repository performance assessment to inform the estimates for Cesium transport parameters, with values solidly based on detailed reactive transport modeling. The presented approach can be used for parameter uncertainty analysis of any complex reactive transport problem and can be applied to analyze any radionuclide of interest.

Linear Polymerization of Protein by Sterically Controlled Enzymatic Cross-Linking with a Tyrosine-Containing Peptide Loop.

The structure of a protein complex needs to be controlled appropriately to maximize its functions. Herein, we report the linear polymerization of bacterial alkaline phosphatase (BAP) through the site-specific cross-linking reaction catalyzed by Trametes sp. laccase (TL). We introduced a peptide loop containing a tyrosine (Y-Loop) to BAP, and the Y-Looped BAP was treated with TL. The Y-Looped BAP formed linear polymers, whereas BAP fused with a C-terminal peptide containing a tyrosine (Y-tag) showed an irregular shape after TL treatment. The sterically confined structure of the Y-Loop could be responsible for the formation of linear BAP polymers. TL-catalyzed copolymerization of Y-Looped BAP and a Y-tagged chimeric antibody-binding protein, pG2pA-Y, resulted in the formation of linear bifunctional protein copolymers that could be employed as protein probes in an enzyme-linked immunosorbent assay (ELISA). Copolymers comprising Y-Looped BAP and pG2pA-Y at a molar ratio of 100:1 exhibited the highest signal in the ELISA with 26- and 20-fold higher than a genetically fused chimeric protein, BAP-pG2pA-Y, and its polymeric form, respectively. This result revealed that the morphology of the copolymers was the most critical feature to improve the functionality of the protein polymers as detection probes, not only for immunoassays but also for other diagnostic applications.