NMR-Based Chemosensing via p-H2 Hyperpolarization: Application to Natural Extracts.

When dealing with trace analysis of complex mixtures, NMR suffers from both low sensitivity and signal overlap. NMR chemosensing, in which the association between an analyte and a receptor is "signaled" by an NMR response, has been proposed as a valuable analytical tool for biofluids and natural extracts. Such chemosensors offer the possibility to simultaneously detect and distinguish different analytes in solution, which makes them particularly suitable for analytical applications on complex mixtures. In this study, we have combined NMR chemosensing with nuclear spin hyperpolarization. This was realized using an iridium complex as a receptor in the presence of parahydrogen: association of the target analytes to the metal center results in approximately 1000-fold enhancement of the NMR response. This amplification allows the detection, identification, and quantification of analytes at low-micromolar concentrations, provided they can weakly associate to the iridium chemosensor. Here, our NMR chemosensing approach was applied to the quantitative determination of several flavor components in methanol extracts of ground coffee.

Structural and thermodynamic signatures that define pseudotriloop RNA hairpins.

Pseudotriloop (PTL) structures in RNAs have been recognized as essential elements in RNA folding and recognition of proteins. PTL structures are derived from hexaloops by formation of a cross-loop base pair leaving a triloop and 3' bulged out residue. Despite their common presence and functional importance, insufficient structural and thermodynamic data are available that can be used to predict formation of PTLs from sequence alone. Using NMR spectroscopy and UV-melting data we established factors that contribute to the formation and stability of PTL structures derived from hepatitis B virus and human foamy virus. The NMR data show that, besides the cross-loop base pair, also a 3' pyrimidine bulge and a G-C loop-closing base pair are primary determinants of PTL formation. By changing the G-C closing base pair into C-G, the PTL switches into a hexaloop. Comparison of these rules with regular triloop hairpins and PTLs from other sources is discussed as well as the conservation of a PTL in human foamy virus and other spumaretroviruses.

Magnetic susceptibility to measure total protein concentration from NMR metabolite spectra: Demonstration on blood plasma.

PURPOSE: Accurate metabolite and protein quantification in blood plasma and other body fluids from one single NMR measurement, allowing for improved quantitative metabolic profiling and better assessment of metabolite-protein interactions. THEORY AND METHODS: The total protein concentration is derived from the common chemical-shift changes-caused by protein-induced bulk magnetic susceptibility (BMS)-measured on well-accessible and exchange-free metabolite resonances. These BMS shifts are simply obtained by external referencing with respect to 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt in a coaxial insert. RESULTS: Based on blood-plasma data from five volunteers, the estimated accuracy of the BMS method is ≤ 5% with respect and comparable to the 3.8% error of the standard colorimetric, Biuret, method. Valine, alanine, glucose, leucine, and lactate display no exchange-induced shift changes. Their well-accessible signals act as reliable probes for pure protein-induced BMS. The slopes and intercepts of their chemical-shift change versus protein concentration were derived from metabolite mixtures with (fatted) human and bovine albumin acting as blood-plasma mimics. CONCLUSION: The BMS method, demonstrated on blood plasma, can also be used on other samples containing sufficient protein (> 10 g/L). Also, it allows measurement of the presence and sign of exchange-induced chemical-shift changes.

2D†NMR Trace Analysis by Continuous Hyperpolarization at High Magnetic Field.

Nuclear magnetic resonance is often the technique of choice in chemical analysis because of its sensitivity to molecular structure, quantitative character, and straightforward sample preparation. However, determination of trace analytes in complex mixtures is generally limited by low sensitivity and extensive signal overlap. Here, we present an approach for continuous hyperpolarization at high magnetic field that is based on signal amplification by reversible exchange (SABRE) and can be straightforwardly incorporated in multidimensional NMR experiments. This method was implemented in a 2D correlation experiment that allows detection and quantification of analytes at nanomolar concentration in complex solutions.

Quantitative trace analysis of complex mixtures using SABRE hyperpolarization.

Signal amplification by reversible exchange (SABRE) is an emerging nuclear spin hyperpolarization technique that strongly enhances NMR signals of small molecules in solution. However, such signal enhancements have never been exploited for concentration determination, as the efficiency of SABRE can strongly vary between different substrates or even between nuclear spins in the same molecule. The first application of SABRE for the quantitative analysis of a complex mixture is now reported. Despite the inherent complexity of the system under investigation, which involves thousands of competing binding equilibria, analytes at concentrations in the low micromolar range could be quantified from single-scan SABRE spectra using a standard-addition approach.

Toward nanomolar detection by NMR through SABRE hyperpolarization.

SABRE is a nuclear spin hyperpolarization technique based on the reversible association of a substrate molecule and para-hydrogen (p-H2) to a metal complex. During the lifetime of such a complex, generally fractions of a second, the spin order of p-H2 is transferred to the nuclear spins of the substrate molecule via a transient scalar coupling network, resulting in strongly enhanced NMR signals. This technique is generally applied at relatively high concentrations (mM), in large excess of substrate with respect to metal complex. Dilution of substrate ligands below stoichiometry results in progressive decrease of signal enhancement, which precludes the direct application of SABRE to the NMR analysis of low concentration (µM) solutions. Here, we show that the efficiency of SABRE at low substrate concentrations can be restored by addition of a suitable coordinating ligand to the solution. The proposed method allowed NMR detection below 1 µM in a single scan.

How a low-fidelity DNA polymerase chooses non-Watson-Crick from Watson-Crick incorporation.

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

Fast production of homogeneous recombinant RNA--towards large-scale production of RNA.

In the past decades, RNA molecules have emerged as important players in numerous cellular processes. To understand these processes at the molecular and atomic level, large amounts of homogeneous RNA are required for structural, biochemical and pharmacological investigations. Such RNAs are generally obtained from laborious and costly in vitro transcriptions or chemical synthesis. In 2007, a recombinant RNA technology has been described for the constitutive production of large amounts of recombinant RNA in Escherichia coli using a tRNA-scaffold approach. We demonstrate a general applicable extension to the described approach by introducing the following improvements: (i) enhanced transcription of large recombinant RNAs by T7 RNA polymerase (high transcription rates, versatile), (ii) efficient and facile excision of the RNA of interest from the tRNA-scaffold by dual cis-acting hammerhead ribozyme mediated cleavage and (iii) rapid purification of the RNA of interest employing anion-exchange chromatography or affinity chromatography followed by denaturing polyacrylamide gel electrophoresis. These improvements in the existing method pave the tRNA-scaffold approach further such that any (non-)structured product RNA of a defined length can cost-efficiently be obtained in (multi-)milligram quantities without in vitro enzymatic manipulations.

Interconverting conformations of slipped-DNA junctions formed by trinucleotide repeats affect repair outcome.

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations.

Nucleic acid helix structure determination from NMR proton chemical shifts.

We present a method for de novo derivation of the three-dimensional helix structure of nucleic acids using non-exchangeable proton chemical shifts as sole source of experimental restraints. The method is called chemical shift de novo structure derivation protocol employing singular value decomposition (CHEOPS) and uses iterative singular value decomposition to optimize the structure in helix parameter space. The correct performance of CHEOPS and its range of application are established via an extensive set of structure derivations using either simulated or experimental chemical shifts as input. The simulated input data are used to assess in a defined manner the effect of errors or limitations in the input data on the derived structures. We find that the RNA helix parameters can be determined with high accuracy. We finally demonstrate via three deposited RNA structures that experimental proton chemical shifts suffice to derive RNA helix structures with high precision and accuracy. CHEOPS provides, subject to further development, new directions for high-resolution NMR structure determination of nucleic acids.

EC-SPE-stripline-NMR analysis of reactive products: a feasibility study.

Flow-through electrochemical conversion (EC) of drug-like molecules was hyphenated to miniaturized nuclear magnetic resonance spectroscopy (NMR) via on-line solid-phase extraction (SPE). After EC of the prominent p38α mitogen-activated protein kinase inhibitor BIRB796 into its reactive products, the SPE step provided preconcentration of the EC products and solvent exchange for NMR analysis. The acquisition of NMR spectra of the mass-limited samples was achieved in a stripline probe with a detection volume of 150 nL offering superior mass sensitivity. This hyphenated EC-SPE-stripline-NMR setup enabled the detection of the reactive products using only minute amounts of substrate. Furthermore, the integration of conversion and detection into one flow setup counteracts incorrect assessments caused by the degradation of reactive products. However, apparent interferences of the NMR magnetic field with the EC, leading to a low product yield, so far demanded relatively long signal averaging. A critical assessment of what is and what is not (yet) possible with this approach is presented, for example in terms of structure elucidation and the estimation of concentrations. Additionally, promising routes for further improvement of EC-SPE-stripline-NMR are discussed.

Chemical shifts in nucleic acids studied by density functional theory calculations and comparison with experiment.

NMR chemical shifts are highly sensitive probes of local molecular conformation and environment and form an important source of structural information. In this study, the relationship between the NMR chemical shifts of nucleic acids and the glycosidic torsion angle, χ, has been investigated for the two commonly occurring sugar conformations. We have calculated by means of DFT the chemical shifts of all atoms in the eight DNA and RNA mono-nucleosides as a function of these two variables. From the DFT calculations, structures and potential energy surfaces were determined by using constrained geometry optimizations at the BP86/TZ2P level of theory. The NMR parameters were subsequently calculated by single-point calculations at the SAOP/TZ2P level of theory. Comparison of the (1)H and (13)C NMR shifts calculated for the mono-nucleosides with the shifts determined by NMR spectroscopy for nucleic acids demonstrates that the theoretical shifts are valuable for the characterization of nucleic acid conformation. For example, a clear distinction can be made between χ angles in the anti and syn domains. Furthermore, a quantitative determination of the χ angle in the syn domain is possible, in particular when (13)C and (1)H chemical shift data are combined. The approximate linear dependence of the C1' shift on the χ angle in the anti domain provides a good estimate of the angle in this region. It is also possible to derive the sugar conformation from the chemical shift information. The DFT calculations reported herein were performed on mono-nucleosides, but examples are also provided to estimate intramolecularly induced shifts as a result of hydrogen bonding, polarization effects, or ring-current effects.

Simultaneous analysis of plasma and CSF by NMR and hierarchical models fusion.

Because cerebrospinal fluid (CSF) is the biofluid which interacts most closely with the central nervous system, it holds promise as a reporter of neurological disease, for example multiple sclerosis (MScl). To characterize the metabolomics profile of neuroinflammatory aspects of this disease we studied an animal model of MScl-experimental autoimmune/allergic encephalomyelitis (EAE). Because CSF also exchanges metabolites with blood via the blood-brain barrier, malfunctions occurring in the CNS may be reflected in the biochemical composition of blood plasma. The combination of blood plasma and CSF provides more complete information about the disease. Both biofluids can be studied by use of NMR spectroscopy. It is then necessary to perform combined analysis of the two different datasets. Mid-level data fusion was therefore applied to blood plasma and CSF datasets. First, relevant information was extracted from each biofluid dataset by use of linear support vector machine recursive feature elimination. The selected variables from each dataset were concatenated for joint analysis by partial least squares discriminant analysis (PLS-DA). The combined metabolomics information from plasma and CSF enables more efficient and reliable discrimination of the onset of EAE. Second, we introduced hierarchical models fusion, in which previously developed PLS-DA models are hierarchically combined. We show that this approach enables neuroinflamed rats (even on the day of onset) to be distinguished from either healthy or peripherally inflamed rats. Moreover, progression of EAE can be investigated because the model separates the onset and peak of the disease.

Quantitative proteomics and metabolomics analysis of normal human cerebrospinal fluid samples.

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.

Fusion of metabolomics and proteomics data for biomarkers discovery: case study on the experimental autoimmune encephalomyelitis.

BACKGROUND: Analysis of Cerebrospinal Fluid (CSF) samples holds great promise to diagnose neurological pathologies and gain insight into the molecular background of these pathologies. Proteomics and metabolomics methods provide invaluable information on the biomolecular content of CSF and thereby on the possible status of the central nervous system, including neurological pathologies. The combined information provides a more complete description of CSF content. Extracting the full combined information requires a combined analysis of different datasets i.e. fusion of the data. RESULTS: A novel fusion method is presented and applied to proteomics and metabolomics data from a pre-clinical model of multiple sclerosis: an Experimental Autoimmune Encephalomyelitis (EAE) model in rats. The method follows a mid-level fusion architecture. The relevant information is extracted per platform using extended canonical variates analysis. The results are subsequently merged in order to be analyzed jointly. We find that the combined proteome and metabolome data allow for the efficient and reliable discrimination between healthy, peripherally inflamed rats, and rats at the onset of the EAE. The predicted accuracy reaches 89% on a test set. The important variables (metabolites and proteins) in this model are known to be linked to EAE and/or multiple sclerosis. CONCLUSIONS: Fusion of proteomics and metabolomics data is possible. The main issues of high-dimensionality and missing values are overcome. The outcome leads to higher accuracy in prediction and more exhaustive description of the disease profile. The biological interpretation of the involved variables validates our fusion approach.

NMR and pattern recognition can distinguish neuroinflammation and peripheral inflammation.

Multiple Sclerosis (MScl) is a neurodegenerative disease of the CNS, associated with chronic neuroinflammation. Cerebrospinal fluid (CSF), being in closest interaction with CNS, was used to profile neuroinflammation to discover disease-specific markers. We used the commonly accepted animal model for the neuroinflammatory aspect of MScl: the experimental autoimmune/allergic encephalomyelitis (EAE). A combination of advanced (1)H NMR spectroscopy and pattern recognition methods was used to establish the metabolic profile of CSF of EAE-affected rats (representing neuroinflammation) and of two control groups (healthy and peripherally inflamed) to detect specific markers for early neuroinflammation. We found that the CSF metabolic profile for neuroinflammation is distinct from healthy and peripheral inflammation and characterized by changes in concentrations of metabolites such as creatine, arginine, and lysine. Using these disease-specific markers, we were able to detect early stage neuroinflammation, with high accuracy in a second independent set of animals. This confirms the predictive value of these markers. These findings from the EAE model may help to develop a molecular diagnosis for the early stage MScl in humans.

The impact of delayed storage on the measured proteome and metabolome of human cerebrospinal fluid.

BACKGROUND: Because cerebrospinal fluid (CSF) is in close contact with diseased areas in neurological disorders, it is an important source of material in the search for molecular biomarkers. However, sample handling for CSF collected from patients in a clinical setting might not always be adequate for use in proteomics and metabolomics studies. METHODS: We left CSF for 0, 30, and 120 min at room temperature immediately after sample collection and centrifugation/removal of cells. At 2 laboratories CSF proteomes were subjected to tryptic digestion and analyzed by use of nano-liquid chromatography (LC) Orbitrap mass spectrometry (MS) and chipLC quadrupole TOF-MS. Metabolome analysis was performed at 3 laboratories by NMR, GC-MS, and LC-MS. Targeted analyses of cystatin C and albumin were performed by LC-tandem MS in the selected reaction monitoring mode. RESULTS: We did not find significant changes in the measured proteome and metabolome of CSF stored at room temperature after centrifugation, except for 2 peptides and 1 metabolite, 2,3,4-trihydroxybutanoic (threonic) acid, of 5780 identified peptides and 93 identified metabolites. A sensitive protein stability marker, cystatin C, was not affected. CONCLUSIONS: The measured proteome and metabolome of centrifuged human CSF is stable at room temperature for up to 2 hours. We cannot exclude, however, that changes undetectable with our current methodology, such as denaturation or proteolysis, might occur because of sample handling conditions. The stability we observed gives laboratory personnel at the collection site sufficient time to aliquot samples before freezing and storage at -80 °C.

Preparation of selective and segmentally labeled single-stranded DNA for NMR by self-primed PCR and asymmetrical endonuclease double digestion.

We demonstrate a new, efficient and easy-to-use method for enzymatic synthesis of (stereo-)specific and segmental (13)C/(15)N/(2)H isotope-labeled single-stranded DNA in amounts sufficient for NMR, based on the highly efficient self-primed PCR. To achieve this, new approaches are introduced and combined. (i) Asymmetric endonuclease double digestion of tandem-repeated PCR product. (ii) T4 DNA ligase mediated ligation of two ssDNA segments. (iii) In vitro dNTP synthesis, consisting of in vitro rNTP synthesis followed by enzymatic stereo-selective reduction of the C2' of the rNTP, and a one-pot add-up synthesis of dTTP from dUTP. The method is demonstrated on two ssDNAs: (i) a 36-nt three-way junction, selectively (13)C(9)/(15)N(3)/(2)H((1',2'',3',4',5',5''))-dC labeled and (ii) a 39-nt triple-repeat three-way junction, selectively (13)C(9)/(15)N(3)/(2)H((1',2'',3',4',5',5''))-dC and (13)C(9)/(15)N(2)/(2)H((1',2'',3',4',5',5''))-dT labeled in segment C20-C39. Their NMR spectra show the spectral simplification, while the stereo-selective (2)H-labeling in the deoxyribose of the dC-residues, straightforwardly provided assignment of their C1'-H2' and C2'-H2' resonances. The labeling protocols can be extended to larger ssDNA molecules and to more than two segments.

Application of fluorine NMR for structure identification of steroids.

Fluorinated steroids were examined using 1D and 2D homo- and heteronuclear (19)F NMR, such as (19)F-(1) H and (19)F-(13)C. The utilization of fluorine NMR accounted for spectral simplification and resulted in a straightforward pathway for the determination of structures including the configuration of these compounds; these steroids present an illustrative example for other types of fluorinated compounds, which are increasingly encountered in drug discovery. The potential of (19)F NMR is elaborated on in detail for two compounds containing diastereotopic fluorines with different coupling patterns. The analysis of the coupling patterns and the through-space interactions resulted in the determination of the structure and configuration. Heteronuclear correlation experiments, i.e. (19)F-(1)H HETCOR, (19)F-(13)C HMQC and HMBC, and (19)F-(1)H HOESY, were applied to determine first the relative stereochemistry and then the molecular configuration at C4 and C5 of a steroidal compound bearing a fused three-membered ring with two fluorine substituents. These examples proved (19)F NMR to be a useful addition to the extensively used (1)H and (13)C NMR within structure elucidation and configuration determination of small molecules.

Enzymatic stereospecific preparation of fluorescent S-adenosyl-L-methionine analogs.

S-Adenosyl-L-methionine (SAM) is the preferred cofactor for biological methyl group transfers to various substrates such as nucleic acids, proteins, and lipids. Here we present stereospecific (>95% of the desired enantiomer) and high-yield preparation of four fluorescent and biologically active SAM analogs and demonstrate their usefulness in binding studies. Using a fluorescence titration experiment, we obtained a K(d) of 0.38 microM for the S-2,6-diaminopurinylmethionine-SAM-III riboswitch complex.