Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems

Dynamic nuclear polarization (DNP) has shown great promise as a tool to enhance the nuclear magnetic resonance signals of proteins in the cellular environment. As sensitivity increases, the ability to select and efficiently polarize a specific macromolecule over the cellular background has become desirable. Herein, we address this need and present a tetrazine-based DNP agent that can be targeted selectively to proteins containing the unnatural amino acid (UAA) norbornene-lysine. This UAA can be introduced efficiently into the cellular milieu by genetic means. Our approach is bio-orthogonal and easily adaptable to any protein of interest. We illustrate the scope of our methodology and investigate the DNP transfer mechanisms in several biological systems. Our results shed light on the complex polarization-transfer pathways in targeted DNP and ultimately pave the way to selective DNP-enhanced NMR spectroscopy in both bacterial and mammalian cells.     

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low‐temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin‐T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long‐range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.     

Nanodisc‐Targeted STD NMR Spectroscopy Reveals Atomic Details of Ligand Binding to Lipid Environments

Saturation transfer difference (STD) NMR spectroscopy is one of the most popular ligand‐based NMR techniques for the study of protein–ligand interactions. This is due to its robustness and the fact that it is focused on the signals of the ligand, without any need for NMR information on the macromolecular target. This technique is most commonly applied to systems involving different types of ligands (e.g., small organic molecules, carbohydrates or lipids) and a protein as the target, in which the latter is selectively saturated. However, only a few examples have been reported where membrane mimetics are the macromolecular binding partners. Here, we have employed STD NMR spectroscopy to investigate the interactions of the neurotransmitter dopamine with mimetics of lipid bilayers, such as nanodiscs, by saturation of the latter. In particular, the interactions between dopamine and model lipid nanodiscs formed either from charged or zwitterionic lipids have been resolved at the atomic level. The results, in agreement with previous isothermal titration calorimetry studies, show that dopamine preferentially binds to negatively charged model membranes, but also provide detailed atomic insights into the mode of interaction of dopamine with membrane mimetics. Our findings provide relevant structural information for the design of lipid‐based drug carriers of dopamine and its structural analogues and are of general applicability to other systems.     

Nondestructive NMR determination of oil composition in transformed canola seeds

Magic-angle spinning (MAS) ¹³C nuclear magnetic resonance (NMR) spectroscopy is a convenient method for nondestructive, quantitative characterization of seed oil composition. We describe results for intact hybrid and transformed canola seeds. The MAS ¹³C NMR technique complements and agrees with gas chromatography results. The spectral resolution approaches that of neat, liquid oils. MAS ¹³C NMR data allow quantitative analysis of major oil components, including saturates and oleic, linoleic, and linolenic acyl chains. ¹³C NMR directly and quantitatively elucidates, triglyceride regiochemistry and acyl chain cis-trans isomers that cannot be quickly detected by other methods. MAS ¹³C NMR can serve as the primary method for development of near-infrared seed oil calibrations. These NMR methods are nondestructive and attractive for plant-breeding programs or other studies (e.g., functional genomics) where loss of seed viability is inconvenient.     

NMR of insensitive nuclei enhanced by dynamic nuclear polarization

Despite the powerful spectroscopic information it provides, Nuclear Magnetic Resonance (NMR) spectroscopy suffers from a lack of sensitivity, especially when dealing with nuclei other than protons. Even though NMR can be applied in a straightforward manner when dealing with abundant protons of organic molecules, it is very challenging to address biomolecules in low concentration and/or many other nuclei of the periodic table that do not provide as intense signals as protons. Dynamic Nuclear Polarization (DNP) is an important technique that provides a way to dramatically increase signal intensities in NMR. It consists in transferring the very high electron spin polarization of paramagnetic centers (usually at low temperature) to the surrounding nuclear spins with appropriate microwave irradiation. DNP can lead to an enhancement of the nuclear spin polarization by up to four orders of magnitude. We present in this article some basic concepts of DNP, describe the DNP apparatus at EPFL, and illustrate the interest of the technique for chemical applications by reporting recent measurements of the kinetics of complexation of 89Y by the DOTAM ligand.     

Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery

Nuclear magnetic resonance (NMR) spectroscopy is a valuable technique for ligand screening, because it exhibits high specificity toward chemical structure and interactions. Dissolution dynamic nuclear polarization (DNP) is a recent advance in NMR methodology that enables the creation of non‐equilibrium spin states, which can dramatically increase NMR sensitivity. Here, the transfer of such spin polarization from hyperpolarized ligand to protein is observed. Mixing hyperpolarized benzamidine with the serine protease trypsin, a “fingerprint” of enhanced protein signals is observed, which shows a different intensity profile than the equilibrium NMR spectrum of the protein, but coincides closely to the frequency profile of a saturation transfer difference (STD) NMR experiment. The DNP experiment benefits from hyperpolarization and enables observation of all frequencies in a single, rapid experiment. Based on these merits, it is an interesting alternative to the widely used STD experiment for identification of protein–ligand interactions.     

Use of magnetic nanoparticles as nanosensors to probe for molecular interactions

Biocompatible magnetic nanosensors have been designed to detect molecular interactions in biological media. Upon target binding, these nanosensors cause changes in the spin-spin relaxation times of neighboring water molecules, which can be detected by magnetic resonance (NMR/MRI) techniques. These magnetic nanosensors have been designed to detect specific mRNA, proteins, enzymatic activity, and pathogens (e.g., virus) with sensitivity in the low femtomole range (0.5-30 fmol).     

Systematic Investigation of Metabolic Oligosaccharide Engineering Efficiency in Intestinal Cells Using a Dibenzocyclooctyne-Monosaccharide Conjugate

Metabolic oligosaccharide engineering (MOE) of cells with synthetic monosaccharides can introduce functionality to the glycans of cell membranes. Unnatural sugars (e. g., peracetylated mannose-azide) can be expressed on the cell surface with the azide group in place. After MOE, the azide group can participate in a copper-free click reaction with an alkyne (e. g., dibenzocyclooctyne, DBCO) probe. This allows the metabolic fate of monosaccharides in cells to be understood. However, in a drug delivery context it is desirable to have azide groups on the probe (e. g. a drug delivery particle) and the alkyne (e. g. DBCO) on the cell surface. Consequently, the labelling efficiency of intestinal cell lines (Caco-2 and HT29-MTX-E12) treated with N-dibenzocyclooctyne-tetra-acetylmannosamine, and the concentration- and time-dependent labelling were determined. Furthermore, the labelling of mucus in HT29-MTX-E12 cells with DBCO was shown. This study highlights the potential for using MOE to target azide-functionalised probes to intestinal tissues for drug delivery applications.     

Foxp3 Silencing with Antisense Oligonucleotide Improves Immunogenicity of an Adjuvanted Recombinant Vaccine against Sporothrix schenckii

Background: In recent years, there has been great interest in developing molecular adjuvants based on antisense oligonucleotides (ASOs) targeting immunosuppressor pathways with inhibitory effects on regulatory T cells (Tregs) to improve immunogenicity and vaccine efficacy. We aim to evaluate the immunostimulating effect of 2′OMe phosphorothioated Foxp3-targeted ASO in an antifungal adjuvanted recombinant vaccine. Methods: The uptake kinetics of Foxp3 ASO, its cytotoxicity and its ability to deplete Tregs were evaluated in murine splenocytes in vitro. Groups of mice were vaccinated with recombinant enolase (Eno) of Sporothix schenckii in Montanide Gel 01 adjuvant alone or in combination with either 1 µg or 8 µg of Foxp3 ASO. The titers of antigen-specific antibody in serum samples from vaccinated mice (male C57BL/6) were determined by ELISA (enzyme-linked immunosorbent assay). Cultured splenocytes from each group were activated in vitro with Eno and the levels of IFN-γ and IL-12 were also measured by ELISA. The results showed that the anti-Eno antibody titer was significantly higher upon addition of 8 µM Foxp3 ASO in the vaccine formulation compared to the standard vaccine without ASO. In vitro and in vivo experiments suggest that Foxp3 ASO enhances specific immune responses by means of Treg depletion during vaccination. Conclusion: Foxp3 ASO significantly enhances immune responses against co-delivered adjuvanted recombinant Eno vaccine and it has the potential to improve vaccine immunogenicity.     

In-cell solid-state NMR as a tool to study proteins in large complexes

A major limitation of solution NMR is molecular tumbling, which is often too slow for detection. Here we demonstrate that solid-state NMR spectroscopy in combination with flash freezing of cells can be used to detect proteins in the cellular environment and provides information on backbone chemical shifts.     

A structural study of epoxidized natural rubber (ENR‐50) ring opening under mild acidic condition

A structural study of ring opening reaction of purified epoxidized natural rubber (ENR) with acetic acid was conducted using the NMR techniques and its thermal characteristic was evaluated with Thermal gravimetry/Differential Thermal Gravimetry (TG/DTG) and Differential Scanning Calorimetry (DSC) analyses. ¹H‐NMR revealed that 19.56% of epoxide was ring‐opened from the total amount of the epoxide unit in ENR‐50 and this was supported by Fourier Transform Infrared (FTIR) spectroscopy. ¹³C‐NMR suggests the fixation of alkyl (R) i.e., acetate group to the epoxide carbon via ester linkage and formation of hydroxyl groups in the polymer chains. The attachment location of R occurred at both most (↑) and least (↓) hindered carbons of the epoxide. The TG/DTG results of acid treated ENR‐50 showed three decomposition steps at 235–338, 338–523, 523–627 °C due to the presence of the polymer chains mixture, i.e., ring‐opened and intact epoxide of ENR‐50. This increases the T_g value of acid treated ENR‐50 at 24.6 °C as compared to purified ENR‐50 at −17.7 °C. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 44123.     

Structure and dynamics of hydrogels and organogels: An NMR spectroscopy approach

Hydrogels and organogels are semi-solid systems, in which a liquid phase is immobilized by a three-dimensional network composed of self-assembled, intertwined polymer/gelator fibers. Investigations pertaining to these systems have only picked up speed in the last few decades. Consequently, many burning questions regarding these systems, such as the specific molecular requirements guaranteeing gelation, still await definite answers. Nonetheless, the application of different hydrogels and organogels to various areas of interest, i.e., as drug delivery devices, has been quick to follow their discoveries.The use of NMR spectroscopy for the characterization of polymer hydrogels and organogels has recently seen enormous growth. The NMR measurements involving magic angle spinning (MAS) in the solid-state NMR, spin relaxation times, nuclear Overhauser enhancements (NOE), or multiple-quantum (MQ) spectroscopy, the pulse field gradient (PFG) technique and magnetic resonance imaging (MRI) allow obtaining the detailed information on morphology, molecular organization, specific interactions and internal mobility of constituents.This review aims at providing a global view and capabilities all of these NMR methods in comprehensive studies of hydrogels and organogels, with special emphasis on the interplay between the morphology and molecular mobility of constituents and the intermolecular interactions.     

Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the ‘four-dimensional’ protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the ‘toolbox’ of mass spectrometry researchers studying higher-order protein structure.     

Synthesis and morphology of carboxylated polyether‐block‐polydimethylsiloxane and the supermolecule self‐assembled from it

In this research, hydroxyl‐terminated polyether‐block‐polydimethylsiloxane (PESO) was synthesized as an intermediate through the hydrosilylation of Si? H‐terminated polydimethylsiloxane with allyl polyoxyethylene polyoxypropylene ether. Then, carboxylated polyether‐block‐polydimethylsiloxane (CPES) was prepared through the reaction of maleic anhydride with PESO. First, the chemical structures of the synthesized polysiloxanes were characterized with IR and ¹H‐NMR spectroscopy, and then the film morphology of CPES and the supermolecule self‐assembled from CPES and N‐β‐aminoethyl‐γ‐aminopropyl polydimethylsiloxane (ASO‐1) was investigated by atomic force microscopy in detail. Experimental results indicated that the superpolysiloxane that self‐assembled from CPES and ASO‐1 showed a film morphology very different from those of CPES and ASO‐1. There were not only many small, bright dots but also some big and marvelous dots circled by dots on the film surface. The morphology of dots circled by dots was estimated to result from aggregates of CPES micelles adsorbed onto the curled ASO‐1 molecule interface. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

ACCELERATED LIGHT-INDUCED AGING OF α-, β-, AND γ-13C-ENRICHED CELL WALL-DEHYDROGENATION POLYMERS STUDIED WITH SOLID STATE 13C NMR SPECTROSCOPY

ABSTRACT

Light-induced aging of lignocellulosic materials has been studied with a new technique involving selectively α-, β-, and γ-¹³C-enriched cell wall-dehydrogenation polymers (CW-DHPs) and solid state ¹³C NMR spectroscopy. The results from cross-polarization magic angle spinning (CP/MAS) ¹³C NMR experiments of unirradiated and irradiated CW-DHP have revealed mainly a decrease in the amount of end-groups of both coniferaldehyde and coniferyl alcohol type. The results suggest that these end-groups become saturated and that the terminal functionalites, i.e., γ-aldehyde and γ-hydroxymethyl groups, at least to some extent, are retained. The results indicate further that no detectable cleavage of the β-O-4 bonds occurs in the examined lignocellulosic model. In terms of proposed mechanisms of yellowing, there is marginal evidence that up to 2% of the α-labeled sites are converted by irradiation to α-carbonyls (aldehyde or ketones); moreover, we cannot dismiss the possibility that the precursor structures giving rise to these few α-carbonyls are β-O-4 structures. The ¹³C-enriched CW-DHP was formed directly on spruce (Picea abies) wood tissue (differentiating xylem) by administering selectively ¹³C-labeled coniferin at pH 6.0 in the presence of glucose oxidase and β-glucosidase, i.e., no phenol-oxidizing enzyme was added and the wood cells’ own enzymes polymerized the precursor.     

Li influx and binding, and li/mg competition in bovine chromaffin cell suspensions as studied by li NMR and fluorescence spectroscopy

Li(+) influx by bovine chromaffin cells, obtained from bovine adrenal medulla, was studied in intact cell suspensions using (7)Li NMR spectroscopy with the shift reagent [Tm(HDOTP)](4-). The influx rate constants, k(i), were determined in the absence and in the presence of two Na(+) membrane transport inhibitors. The values obtained indicate that both voltage sensitive Na(+) channels and (Na(+)/K(+))-ATPase play an important role in Li(+) uptake by these cells. (7)Li NMR T(1) and T(2) relaxation times for intracellular Li(+) in bovine chromaffin cells provided a T(1)/T(2) ratio of 305, showing that Li(+) is highly, immobilized due to strong binding to intracellular structures. Using fluorescence spectroscopy and the Mg(2+) fluorescent probe, furaptra, the free intracellular Mg(2+) concentration in the bovine chromaffin cells incubated with 15 mM LiCl was found to increase by about mM after the intracellular Li(+) concentration reached a steady state. Therefore, once inside the cell, Li(+) is able to displace Mg(2+) from its binding sites.     

Specificity of a UDP‐GalNAc Pyranose–Furanose Mutase: A Potential Therapeutic Target for Campylobacter jejuni Infections

Pyranose–furanose mutases are essential enzymes in the life cycle of a number of microorganisms, but are absent in mammalian systems, and hence represent novel targets for drug development. To date, all such mutases show preferential recognition of a single substrate (e.g., UDP‐Gal). We report here the detailed structural characterization of the first bifunctional pyranose–furanose mutase, which recognizes both UDP‐Gal and UDP‐GalNAc. The enzyme under investigation (cjUNGM) is involved in the biosynthesis of capsular polysaccharides (CPSs) in Campylobacter jejuni 11168. These CPSs are known virulence factors that are required for adhesion and invasion of human epithelial cells. Using a combination of UV/visible spectroscopy, X‐ray crystallography, saturation transfer difference NMR spectroscopy, molecular dynamics and CORCEMA‐ST calculations, we have characterized the binding of the enzyme to both UDP‐Galp and UDP‐GalpNAc, and compared these interactions with those of a homologous monofunctional mutase enzyme from E. coli (ecUGM). These studies reveal that two arginines in cjUNGM, Arg59 and Arg168, play critical roles in the catalytic mechanism of the enzyme and in controlling its specificity to ultimately lead to a GalfNAc‐containing CPS. In ecUGM, these arginines are replaced with histidine and lysine, respectively, and this results in an enzyme that is selective for UDP‐Gal. We propose that these changes in amino acids allow C. jejuni 11168 to produce suitable quantities of the sugar nucleotide substrate required for the assembly of a CPS containing GalfNAc, which is essential for viability.     

Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm,Eisenia andrei

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.