WO2022238350A1 - Biocapteur dnp - Google Patents

Biocapteur dnp Download PDF

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Publication number
WO2022238350A1
WO2022238350A1 PCT/EP2022/062531 EP2022062531W WO2022238350A1 WO 2022238350 A1 WO2022238350 A1 WO 2022238350A1 EP 2022062531 W EP2022062531 W EP 2022062531W WO 2022238350 A1 WO2022238350 A1 WO 2022238350A1
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electron
spin
measurement
ligand
solution
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PCT/EP2022/062531
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English (en)
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Jonas MILANI
Felipe SAENZ
Mika TAMSKI
Jean-Philippe Ansermet
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Ecole Polytechnique Federale De Lausanne (Epfl)
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
    • G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
    • G01N24/088—Assessment or manipulation of a chemical or biochemical reaction, e.g. verification whether a chemical reaction occurred or whether a ligand binds to a receptor in drug screening or assessing reaction kinetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/62—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using double resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/28—Details of apparatus provided for in groups G01R33/44 - G01R33/64
    • G01R33/282—Means specially adapted for hyperpolarisation or for hyperpolarised contrast agents, e.g. for the generation of hyperpolarised gases using optical pumping cells, for storing hyperpolarised contrast agents or for the determination of the polarisation of a hyperpolarised contrast agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46—NMR spectroscopy

Definitions

  • the present invention relates to a method for sensitive and selective quantitative detection of biomolecules in liquid state as well as to assay reagents for use in such methods and uses of such assay reagents in such methods.
  • Biological diagnostics such as antibodies detection or drug screening play a significant role in modern medicine or discovery of new pharmaceutical active molecule, but also generally in the medical field, e.g. for diagnostic purposes.
  • Time of analysis, level of detection, quantity of reactive material and cost are important parameters for their efficiency and widespread suitability.
  • New methods have been developed based on different principles including magnetic nuclear resonance, see e.g. WO-A-2009045551.
  • Nuclear Magnetic Resonance (NMR) spectroscopy has significant advantages like the biocompatibility but suffers of its low sensitivity.
  • DNP Dynamic Nuclear Polarization
  • the DNP agents can have the structure A-X-L-R, where A is none or an amphiphilic group; X is a coupling group capable of site-specific binding with the analyte or, when A is an amphiphilic group, capable of site-specific binding with the amphiphilic group; L is a bond or a linker group; and R is a poly-radical group.
  • the methods of measurement of an analyte comprising an NMR-detectable nucleus include the steps of providing a frozen sample containing the analyte and a DNP agent; applying radiation having a frequency that excites electron spin transitions in the DNP agent at an intensity to polarize the NMR- detectable nucleus; and detecting a signal from nuclear spin transitions in the NMR- detectable nucleus.
  • WO-A-03091700 proposes NMR methods for mapping or otherwise identifying the amino acid sequence and conformation of a portion of a protein that is involved in ligand binding. 2
  • the method finds utility in the process of elucidating the amino acid sequence and conformation of an epitope of, for example, an antigen or an antibody that binds to the antigen.
  • WO-A-0040988 proposes an in vitro d-DNP assay method which comprises the use of an assay reagent containing at least one NMR active nucleus, and hyperpolarizing at least one NMR active nucleus of the assay reagent; and analyzing the assay reagent and/or the assay by NMR spectroscopy and/or NMR imaging.
  • the assay reagent may contain an artificially high concentration of an NMR active nucleus.
  • NMR nuclear magnetic resonance
  • diagnostic techniques based on NMR have been developed but generally suffer from its inherently low sensitivity.
  • only a small fraction of the available nuclear spins (circa 10 5 ) can be polarized at room temperature, thus requiring particular conditions or long acquisition times in order to obtain satisfactory signal intensities.
  • hyperpolarization by dynamic nuclear polarization (DNP) has recently attracted interest in order to circumvent the problem.
  • DNP first introduced by Albert Overhauser in 1953, is a physical process where the nuclear spin polarization of a substance of interest is increased above its thermal equilibrium by transferring the electron spin polarization from a polarizing agent (e.g.
  • a radical to nuclei upon microwave irradiation, thus allowing to enhance the substance’s NMR signals over measurement.
  • Hyperpolarization mechanisms depend on different parameters such as temperature, phase of matter, magnetic field, molecular tumbling rate and spin relaxation processes. Since its initial report in 2003, dissolution-DNP (d-DNP) was shown to be one of the most efficient methods to hyperpolarize a substance, where enhancements of up to 5 orders of magnitude can be reached.
  • the polarization step requires severely constrained conditions, as it consists in the transfer of polarization from radicals to nuclei at high magnetic fields (> 3 T) and low temperatures ( ⁇ 4 K) in an amorphous solid, followed by rapid dissolution in a suitable hot solvent to obtain a hyperpolarized solution.
  • the solution can subsequently be measured or used for biological analysis, such as to study ligand- receptor interactions, or enzyme kinetics.
  • dissolution-DNP Overhauser-DNP
  • O-DNP is less constraining since the process can occur under ambient conditions, low magnetic fields ( ⁇ 2 T) and in the liquid state, although the enhancement of the NMR signals is relatively modest in comparison.
  • direct hyperpolarization of liquids can be readily performed with accessible equipment and Overhauser-DNP devices can be miniaturized and integrated to conventional NMR spectrometers.
  • various 3 potential applications of DNP rely on the NMR contrast produced rather than the magnitude of the signal enhancement, and Overhauser-DNP still allows substantial signal gains for the improvement of sensitivity and acquisition time.
  • Overhauser-DNP is a more modest method then the above-mentioned d-DNP but is much less constraining. Setup is very simple and very cheap in comparison with d-DNP and allows the use of a liquid phase during the whole of the measurement. So it can be carried out under ambient conditions.
  • the method proposed here does not use DNP just as a signal amplifier but uses the DNP mechanisms in liquid to determine presence or absence of protein-ligand interaction or more generally speaking, a change in the molecular tumbling of the system of interest which in turn allows to draw conclusions on the status of this system of interest or the presence of the system of interest, preferably quantitatively.
  • Biological diagnostic such as antibodies detection or drug screening play a significant role in modern medicine or discovery of new pharmaceutical active molecules.
  • New methods are presented based on different principles including nuclear magnetic resonance (NMR).
  • NMR spectroscopy has significant advantages like biocompatibility, but suffers of its low sensitivity.
  • Dynamic nuclear polarization proposes to overpass this problem.
  • Overhauser-DNP (O-DNP) is a modest method but requires much less experimental overhead than dissolution-DNP or MAS-DNP (Magic angle spinning-Dynamic nuclear polarization). Since it requires a liquid phase, it can be achieved at ambient condition. The method allows to quantify proteins in solution by comparing the O-DNP signal of a solution with different concentrations of protein.
  • the biochemical system composed by avidin with the spin labelled ligand biotin-TEMPO was taken as proof of concept example with cryo-EM and XRD. Visualizing the structure allowed to measure the degree of liberty of free radical and water accessibility. Moreover, a 10 A distance between two TEMPO in the complex avidin/biotin-TEMPO supposed non-trivial effect like electron-electron coupling.
  • Overhauser-DNP is inter alia reported to detect and quantify receptor-ligand complexes in solution.
  • a radical moiety attached to an adequate ligand acts as the molecular probe, hereafter referred to as the polarizing ligand.
  • the presence of the polarizing ligand and a corresponding receptor analyte results in the formation of a molecular complex, and, provided that the receptor is sufficiently large, 4 hinders the motion (i.e. tumbling rate) of the radical center.
  • the reduced tumbling increases the magnetic anisotropy (i.e.
  • This method is particularly suitable in the case of a total immobilization of the radical in a binding pocket in the protein like with avidin. If the radical is less affected by the tumbling rate as in the case of a binding site on the surface of a protein, it is still advantageous to use the method.
  • the present invention therefore proposes a method as defined in the appended claims, as well as the chemicals as required in such a method and the use of these chemicals in such a method.
  • Hyperpolarization is a physical process in which the nuclear spin polarization in a material of interest is increased above its thermal equilibrium value, thus allowing enhancing the nuclear magnetic resonance (NMR) signals.
  • Hyperpolarization mechanisms depend on a number of different parameters such as temperature, state of matter, magnetic field, molecular properties, time correlation, spin relaxation and spin-spin coupling.
  • the proposed invention here comprises to take advantage of molecular tumbling to identify, characterize and/or even quantify a substance of interest (e.g. a polymer, protein, macromolecule or supramolecular assemblies) in solution.
  • a substance of interest e.g. a polymer, protein, macromolecule or supramolecular assemblies
  • this method it will be demonstrated that it is possible to use this method to e.g. quantify the concentration of the protein avidin as proof of concept.
  • a spin-labeled molecule capable of achieving dynamic nuclear polarization by nuclear Overhauser effect (O-DNP) is e.g. attached to a linker.
  • the presence of a molecule binding the linker, such as avidin will create clusters or aggregates or even a new entity with chemical bonds linking to the target, thus strongly affecting the NOE dynamics (e.g. the correlation time and/or electron - electron coupling) in correlation with the concentration.
  • NOE dynamics e.g. the correlation time and/or electron - electron coupling
  • the advantage of this method is double because the presence of the molecule of interest acts directly on the hyperpolarization mechanism.
  • the one-step method introduced herein greatly simplifies the detection of the analyte 5 compared e.g. to a standard immunological assay measurement, which involves different reactants and several steps. It is not equivalent to the dissolution DNP (d-DNP) which is a two steps method (hyperpolarization in frozen state and analysis in liquid state).
  • d-DNP dissolution DNP
  • the present method does not include the measurement of frozen samples.
  • the present method is exclusively working in the liquid state, i.e. in solution.
  • Immunoassay tests (see Fig. 3, left flow diagram) usually rely on optical detection, which requires the removal of excess fluorophores. Therefore, in order to rinse the system, the antigen is bound to a surface. This is commonly done in a few steps as explained in the diagram.
  • the proposed method according to one specific embodiment as illustrated in Fig. 3, right diagram does not require rinsing, as the linker can be free in solution and the excess does not need to be removed.
  • the detection is directly performed after the addition of the antibody thus allowing the measurement in a microfluidic system or in a batch reactor.
  • the intensity of the magnetic field is usually a bottleneck for hyperpolarization technologies, as superconducting magnets are very expensive.
  • the proposed method only requires low static magnetic fields in the range of 0.3 T, which can be generated with cheap, permanent magnets, thus allowing the use of existing commercial solutions.
  • the proposed method is at the intersection of quantum mechanics and biotechnology.
  • the principles applied here require a deep understanding of both fields.
  • the effect was predicted by examining the quantum mechanical theory describing the Overhauser DNP, and then the adequate biochemical system in order to verify the prediction were designed.
  • the spin-labels were functionalized with modern organic chemistry techniques.
  • the present invention proposes the following method:
  • a method for detecting a molecule, or in terms of the physical basis a change in molecular tumbling and/or electron spin-spin coupling of an electron-spin labelled system in solution wherein nuclear spins of said electron-spin labelled system (and/or of a linker already connected with said electron-spin labelled system) and/or nuclear spins of the solvent of the electron-spin labelled system are measured in solution using Overhauser dynamic 6 nuclear polarization in a first reference measurement. So the nuclear spins of the entire solution are preferably measured.
  • nuclear spins of said electron-spin labelled system (and/or of the linker already connected with said electron-spin labelled system) and of at least one further molecule aggregated and/or (chemically or physically) linked with at least one of said electron-spin labelled systems (and if present a linker already connected therewith) and not present in said first measurement and/or nuclear spins of the solvent of the aggregate are measured in solution using Overhauser dynamic nuclear polarization in a second measurement.
  • the nuclear spins of said electron-spin labelled systems and/or nuclear spins of the solvent of the system are measured under different conditions or in a different environment than in the first measurement in solution using Overhauser dynamic nuclear polarization in a second measurement.
  • a difference in Overhauser dynamic nuclear polarization enhancement spectrum (can be in both directions, enhancement of second measurement relative to first measurement or the other way round) between said first and second measurements is taken as a measure of the change in molecular tumbling and/or electron spin-spin coupling of said electron-spin labelled system and/or of the presence or form of an aggregate of said at least one further molecule aggregated and/or (chemically or physically) linked with at least one of said electron-spin labelled systems.
  • the change in molecular tumbling and/or electron spin-spin coupling and more specifically e.g. the quantity of a detected molecule or e.g. the conformation thereof is derived from the measured Overhauser dynamic nuclear polarization enhancement spectrum.
  • the studied effects can affect not only the on-resonance points but also different frequencies. That influence depends on free radical nature, solvent, temperature, concentration of free radical.
  • the full integral or partial integral or mathematical combination of several points of the Overhauser dynamic nuclear polarization enhancement spectrum can be used in a manner to increase the sensibility and/or decrease the error on the measurement.
  • the numerical evaluation of the difference for the determination of a change in molecular tumbling and/or electron spin-spin coupling can take the form of integrating the full spectrum of the first measurement and integrating the full spectrum of the second measurement and taking the difference. It may however also take the form of integrating only parts of the full spectrum, i.e. a certain window around the main detection peak in the first and the second measurement and taking the difference thereof. Alternatively, it may take the form of selecting single amplitudes at specific frequencies in the first and the second measurement and forming the difference. Also combinations thereof possible, as well as forming sums and differences.
  • the method is proposed based on the Overhauser Dynamic Nuclear Polarization (O-DNP) in liquid.
  • O-DNP Overhauser Dynamic Nuclear Polarization
  • the Overhauser effect for a two 1 ⁇ 2 spin system in the absence of g-anisotropy for the Electron Paramagnetic Resonance (EPR) and zero field splitting is describes by (1): with leakage factor f, saturation factor s , g 5 and g, the gyromagnetic ratio of the electron and nuclei, respectively.
  • the coupling factor x is given by (2): with f M the molar fraction between nuclei bound with electron, so with hyperfine coupling, and free nuclei, without hyperfine coupling.
  • the paramagnetic contribution to the nuclear spin relaxation R lp is (3): wherein w, and ⁇ % are the nuclei and electron resonance frequencies, respectively.
  • T C is the correlation time for dipole-dipole interaction k and k dlff are related to transition probabilities.
  • the spectral density function ] ⁇ w,t) is defined as follows (4): with t as the correlation time and w the resonance frequency with A the contact hyperfine coupling constant, r con the time correlation for the contact interaction and S the unpaired electron’s spin.
  • k is the constant for the transition probabilities between the electron-nucleus spin state in the four-level energy system for the case of non-diffusion controlled relaxation (6): 8 with m 0 vacuum permeability, g e the electron g-factor, m B Bohr magneton constant and r the electron-nuclei distance.
  • k dl ff j s the constant for transition probabilities for the case of diffusion controlled relaxation (7): 1320007G 2 N A [M] Y fg ⁇ B 2 S(S + 1)
  • D M is the diffusion coefficient of the molecules bearing the unpaired electron and D L the diffusion coefficient of the molecule with the nucleus.
  • [M] is the molar concentration of the paramagnetic moiety and d is the distance of closest approach between unpaired electron(s) and nucleus.
  • the enhancement of polarization transfer during O-DNP is therefore a function of the correlation time and of the distance between spins.
  • a more elaborate theory may include anisotropy of the EPR line and multi electrons interaction but still will be along the same main principles.
  • a system which changes its correlation time and/or the spin-spin interaction will change its O-DNP enhancement, as well, and it does this in a way which can be used for quantitative analysis and not just for identification or characterization if using ligands having high selectivity and sensitivity for attaching to a corresponding receptor.
  • Fig. 5 shows that not simply the NMR intensity, but also the DNP spectrum, changes in a drastic faction when the tumbling rate changes, due to molecular complexes formation.
  • the detection can be done therefore by measuring one point on the DNP spectrum or by comparing different points on the DNP spectra, i.e the shape of the spectra. This is the basis of this invention as is studied, as proof of concept, in the context of the biological self- assembly effect on the DNP enhancement with a TEMPO-based radical.
  • the difference in enhancement spectrum between said first and second measurements is used for qualitative and/or quantitative analysis of at least one of the following:
  • Said electron-spin labelled system can be an organic paramagnetic molecule, preferably one which is soluble in water or in a solvent of interest, which is complexed or chemically linked to a ligand (e.g. an antibody or antigen) having a selective complexing and/or aggregating behaviour with a biomolecular structure.
  • a ligand e.g. an antibody or antigen
  • the electron-spin labelled system is measured complexed or chemically linked to said ligand
  • the electro-spin labelled system complexed or chemically linked to said ligand is added to a solution of said biomolecular structure upon formation of chemical links, aggregates or complexes with said biomolecular structure.
  • the corresponding aggregates have a significantly larger correlation time of the molecular tumbling due to the increased size correspondingly leading to a significant change in the DNP enhancement, which can be used for identification and quantification purposes. Due to the fact that only the aggregated electro-spin labels will lead to an enhancement, this can be used for quantitative analysis, if need be compensation can be used for the signal stemming from non-bound electro-spin labels.
  • the difference in Overhauser dynamic nuclear polarization enhancement between said first and second measurements can be taken as a measure of the presence of said biomolecular structure, preferably in a quantitative manner.
  • Quantitative analysis of samples with unknown concentration of said biomolecular structure can be made available by calibration with at least one series of second measurements of reference samples having known concentration of said biomolecular structure and comparing the enhancement or a combination of several enhancements at different 10 microwave frequencies of the unknown sample with the enhancements in this calibration curve.
  • double and/or multiple ligand structures can be used to provoke the formation of aggregates involving several of said biomolecular structures in the second measurement.
  • the formation of these larger aggregates leads to even more pronounced slowing down of the molecular tumbling and correspondingly to even more significant change in the enhancement properties in the DNP experiment.
  • the ligands attached to the electro-spin labelled system do not have to be the same ligands as the ligands for double and/or multiple ligand structures, in fact using different ligands can be used to tailor the experimental conditions and to lead to even more selectivity and/or sensitivity.
  • Said electron-spin labelled system can generally be an organic paramagnetic molecule and this organic paramagnetic molecule can be chemically linked or aggregated with an antigen or an antibody as a linker.
  • Organic paramagnetic molecules according to this invention comprise systems such as nitroxyl radicals, such as but not limited to TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl), di-tert-butyl nitroxide, PROXYL (2,2,5,5-Tetramethylpyrrolidine-N-oxyl nitroxide), BDPA (Bisdiphenylenephenylallyl), Trityl (Triphenylmethyl), Methyl viologen (1,T-Dimethyl-4,4 - bipyridinium), DPPH (2,2-Diphenyl-1-picrylhydrazyl), triarylammonium radical, Blatter’s radicals (Benzo[e][1,2,4]triazinyl) or combinations thereof. Also possible are organometallic systems with a paramagnetic metallic centre.
  • the DNP measurements can take place at a static magnetic field strength in the range of 0.1-1 T, preferably in the range of 0.2-0.6 T, preferably using microwave irradiation at the respective appropriate irradiation frequency with an irradiation power in the range of 0.01 - 10 W, preferably in the range of 1 - 10 W, preferably for a duration in the range of 0.1 - 60 s, more preferably in the range of 0.5 - 10 s.
  • Said measured nuclear spins are normally selected from the group consisting of 1 H, 13C, 15N, 170, 29Si, 31P.
  • Preferably said nuclear spin is 1 H.
  • Said difference in Overhauser dynamic nuclear polarization enhancement between said first and second measurements can preferably be used for the identification or quantitative identification of biomolecules selected from the group consisting of: peptides and/or proteins, including antibodies, RNA, DNA, lipids, fatty acids, glycolipids, saccharides, vitamins, hormones, metabolites, lignin, and combinations or aggregates thereof, including viruses or microorganisms including bacteria, eukaryotes including fungi.
  • biomolecules selected from the group consisting of: peptides and/or proteins, including antibodies, RNA, DNA, lipids, fatty acids, glycolipids, saccharides, vitamins, hormones, metabolites, lignin, and combinations or aggregates thereof, including viruses or microorganisms including bacteria, eukaryotes including fungi.
  • the present invention relates to an electron-spin labelled system in the form 11 of an organic paramagnetic molecule (e.g. TEMPO for any of the above-mentioned systems) and wherein this organic paramagnetic molecule is chemically linked or aggregated with an antigen or an antibody as a linker for use in a method as detailed above. Furthermore the present invention relates to the use of such an electron-spin labelled system as detailed above in a method as given above.
  • an organic paramagnetic molecule e.g. TEMPO for any of the above-mentioned systems
  • this organic paramagnetic molecule is chemically linked or aggregated with an antigen or an antibody as a linker for use in a method as detailed above.
  • Fig. 1 shows a schematic representation of the self-assembly system, wherein a solution with radical-ligand molecules free in solution (upper left) is mixed with a solution with a counter-ligand such as a biomolecule, e.g. a protein (upper right), and wherein upon mixing (lower illustration) a cluster or complex formation is provoked, and where clusters change rotational motion and distance between free radicals;
  • a solution with radical-ligand molecules free in solution (upper left)
  • a counter-ligand such as a biomolecule, e.g. a protein (upper right)
  • Fig. 2 shows a schematic representation of the self-assembly system, wherein solution A is a solution with radical-ligand molecule free in solution and with double or multiple ligands; solution B is a solution with a counter-ligand such as biomolecule or protein; solution C illustrates the mixing of A and B provoking a self-assembly of clusters, and where clusters change rotational motion and distance between free radicals;
  • Fig. 3 shows in a schematic representation a classical immunological assay (left flow diagram) as opposed to the proposed DNP biosensor assay;
  • Fig. 4 shows 1 H NMR spectra at 13.5 MHz with and without microwave irradiation at 8.884 GHz (a) and 8.931 GHz (b) at a power of 0.13 W with 1 s of irradiation and for sample with and without linked target;
  • Fig. 5 shows DNP spectra of solutions (integral of NMR signal) without linked target and with linked target as a function of the MW irradiation frequency
  • Fig. 6 shows a possible calibration curve based on signals obtained at different microwave irradiation frequencies
  • Fig. 7 shows schematic representations of further variants of the self-assembly system
  • Fig. 8 shows schematic representations of yet further variants of the proposed method
  • Fig. 9 shows the structure characterization of the saturated avidin bound to four biotin- TEMPO ligands
  • A Electron density determined by cryo-EM
  • B Ribbon diagram showing the four polarizing ligands (darker shading, arrows)
  • C Side view of the surface structure with two protruding polarizing ligands (darker shading, arrows)
  • D Overview of the saturated avidin showing a single receptor subunit (darker shading, dashed arrow) containing a ligand (darker shading, solid arrow);
  • E Sticks model of the vicinity of nitroxide radical with distance with water molecule and main polar structures;
  • F Molecular structure of the polarizing ligand Biotin-TEMPO;
  • Fig. 10 shows O-DNP spectra of the reference biotin-TEMPO solution (solid arrows) and the avidin-biotin-TEMPO complex solution (dashed arrows) with a microwave irradiation power of 0.1 W (dashed lines) and 0.3 W (solid lines).
  • 1H-NMR spectra under microwave irradiation 0.3 W, solid and dashed arrow lines
  • without microwave dotted arrow lines
  • Fig. 11 shows in A: an O-DNP scan for concentration from 1.5625 uM to 200 uM of Avidin in a constant concentration of 800 uM of Biotin-TEMPO; MW irradiation power is equal to 0.1 W, irradiation time is 1 s, 5 scans per measurement and a cooling time of 10 second between each scan; B: calculated R value from equation 1 for each sample with peak A corresponds to the integral of NMR peak at the MW frequency of 8.884 GHz and peak B corresponds to the integral of NMR peak at the MW frequency of 8.931 GHz; each point is made with a total of 10 scans;
  • Fig. 12 shows a structure characterization of CRP saturated with phosphocholine ligands
  • A Overview of the saturated CRP showing a single receptor subunit (arrow).
  • B Ribbon diagram showing the five phosphocholine ligands (solid arrows) and the Ca ions (dashed arrows).
  • C Side view of the surface structure with the phosphocholine ligands (arrows);
  • D Sticks model of the binding pocket containing a phosphocholine ligand bound to two Ca ions;
  • E Molecular structure of the polarizing ligand phosphonooxy-TEMPO;
  • Fig. 1 illustrates the general principle of the method schematically including the preferably required building blocks.
  • a free radical 1 is required to provide for the unpaired electron necessary for the Overhauser dynamic nuclear polarization effect.
  • the nuclear spins typically the protons in the solvent or the protons of the corresponding organic or metallic organic systems involved, which is also required for the method since it is the nuclear spins also of the solvent, which are measured.
  • the free radical 1 is linked, chemically by chemical bond and/or physically, for example by complexing to a ligand 2, which acts as a highly selective moiety to only attach with a high association constant to a very specific counter-moiety.
  • a ligand 2 acts as a highly selective moiety to only attach with a high association constant to a very specific counter-moiety.
  • the idea of this ligand 2 is to attach highly selectively and specifically the free radical to the actual target system, in this case designated as counter ligand 3.
  • the counter ligand 3 can have, as in this illustration, several such selective binding sites for the ligand, but it can also have only one selective binding site. The more binding sites the more ligands and correspondingly the more free radicals will be attached to the corresponding counter ligand 3 to form the actual complex 6, if the free radical covalently linked with the ligand 4, provided in solution (upper left) is added to a solution with the counter ligand 3 (upper right) and mixed.
  • the degree of DNP enhancement can also be used for the determination of an association/disassociation constant for example if the total concentration of the counter ligand 3 in the solution is known and the association/disassociation properties are to be analysed.
  • the DNP enhancement effect will be driven by the fact that in the first measurement, which basically corresponds to a measurement of the upper left solution illustrated in this figure, so without the counter ligand 3, the molecular tumbling and the corresponding correlation time will be much shorter as the counter free radical/ligand 4 is much smaller, than the molecular tumbling correlation time of the aggregate, which is much larger. Any non-linearity of the experimental of the enhancement and/or of the enhancement due to the change in molecular tumbling can be taken care of by corresponding calibration curves.
  • Fig. 2 shows a further development and further preferred variant of the proposed method.
  • the DNP enhancement is a function of the correlation time at the electron resonance frequency.
  • empiric measurement shows a rather high frequency dependence as shown in Fig. 5.
  • Rotational tumbling constants for proteins in water at room temperature are typically in the range of nanoseconds and the smaller molecules formed by the spin labelled molecule covalently linked with the ligand have shorter correlation times typically in the range of picosecond. Therefore the enhancement effect is highly dependent on the correlation time in the window of interest.
  • TEMPO-Biotin is dissolved in PBS to create 20 mM solution (solution A).
  • Avidin is dissolved in PBS to create a 10 mM solution (solution B).
  • Solution A, solution B and PBS are mixed to create ten solutions with a constant concentration of TEMPO-Biotin of 800 microM and concentration from 0 to 200 microM of avidin.
  • Fig. 4 shows 1H NMR spectra at 13.5 MHz with and without microwave irradiation at 8.883 GHz (a) and 8.930 GHz (b) at a power of 0.3 W with 1 s of irradiation for sample with (solution A) and without linked target (solution B).
  • Fig. 5 shows DNP spectra of solutions (integral of NMR signal) without linked target and with linked target as a function of the MW irradiation frequency.
  • the solution is made with 800 microM of DNP active molecule, biotin-TEMPO with 200 microM or without avidin.
  • the spectra represented by solid lines were performed with 0.3 W of power and 1 s of irradiation time with 120 s of cooling time. Dashed line experiments were performed with 0.1 W of power and 1 s of irradiation time with 10 s of cooling time. Each measurement was done with 5 scans
  • Fig. 6 shows a possible calibration curve based on signals obtained at different microwave irradiation frequencies at 8.883 GHz and 8.930 GHz.
  • Nine samples with avidin concentration from 1.562 microM to 200 microM were studied with 2x5 scans (5 scans for one given frequency). Error bars are based on signal-to-noise in NMR spectra.
  • the detection uses a non-zero spin such as 1 ⁇ 2 spin.
  • the spin of detection can be in the non reactive system (solvent, non-reactive molecules) or in the reactive system ((electron) spin label, ligand, receptor et cetera).
  • the non-reactive system is comprises the solvent and molecules which are not involved in the interaction system.
  • the reactive system comprises or rather consists of the molecule which is detected (molecule of interest (MOI)) and other molecules with which MOI has a chemical interaction including the ligand and the spin label.
  • MOI molecule of interest
  • the chemical interaction leading to the complexing can be irreversible or reversible.
  • the reactive system can be in two states: states with MOI and state without MOI.
  • the global system is the sum of non-reactive and reactive system.
  • the illustrations in Fig. 7 and 8 represent schematically possible reactive systems involving these two different states leading to differential DNP enhancement behaviour that can be used for detection and characterisation. The two states are separated by arrows.
  • the principle of the measurement is that the Overhauser DNP response is different between the two states.
  • the measurement comprises an excitation with microwave and a measurement with an NMR detection system.
  • the energy of excitation is injected in the global system.
  • the two sub-systems interact each other.
  • the final response can come from the global system or from one of the sub-systems.
  • spin labelled designates a non-zero spin such as 1 ⁇ 2 spin. At least one type of free radical with unpaired electron is present in the global system, whether in the non reactive system and/or reactive system.
  • catalyst used in these figures includes enzymes.
  • auxiliary used in these figures includes biological cofactors.
  • a receptor 11 with single or multiple binding sites is exposed to the free radical/ligand system 4 as opposed to exposing it at the same time also to an auxiliary such as a biological cofactor.
  • the proposed method in such a situation can be used to identify the importance and also the effect of a cofactor or any other system directly or indirectly influencing the disassociation/association constant of the ligand to the receptor.
  • the free radical i.e. the electron spin labelled molecule is attached to a receptor 13 with single or multiple 16 binding sites.
  • this receptor 13 acts as the ligand discussed above attached to the free radical.
  • the idea of this experiment is to use the proposed method to determine the association/disassociation behaviour of this receptor 13 with the spin label with a complementary receptor 14 with single or multiple binding sites, and to determine the association/disassociation behaviour as a function of the presence of a ligand 20, which is required for the binding, so which is a double ligand for both receptors 13 and 14.
  • the proposed method is used for monitoring a catalytic chemical reaction between a free radical 1 and a receptor 15 with single or multiple binding sites, where the attachment is catalysed by the catalyst 17.
  • Using the proposed method allows monitoring the change in polarization enhancement when measuring the free radical 1 alone compared with measuring the reaction including reagents where the receptor 15 is catalytically bound to the free radical 1.
  • Fig. 8 shows uses of the proposed method without analysing specific receptors or counter ligands.
  • the spin label 1 is combined with a ligand to form the moiety 4, however in this case the ligand is not actually a ligand but rather a monomer or oligomer (for chemical reactions) or a system allowing for physicochemical attachment to same systems.
  • the spin label can be used to study, by way of the proposed method, the behaviour of for example a polymer optimisation 18 driven by a catalyst 17 to form the oligomer or polymer, and the corresponding back reaction in the form of degradation.
  • the method is applied to the situation where the free radical is attached to a molecular chain at both ends or just at two different positions, also possible is the presence of only one such free radical (not illustrated) and this system is subjected to 17 further molecule 22 which acts as a nucleation core or a nucleation ligand, for example this can be used to monitor the folding of an organometallic bio system around the metal centre.
  • the free and unfolded protein can be joined to a free radical spin label 1 , this can be measured in the reference experiment, and then the metal centre can be added and the folding can be monitored since the molecular tumbling will be a function of this folding around the metal centre.
  • the situation is shown where there is no particular molecular system to induce the change in molecular confirmation of the chain which is attached to a spin label.
  • the change in molecular confirmation in this case can for example be initiated by a change in a salt concentration, by change in a temperature or the like, and the proposed method can be used for monitoring the folding or for determining the equilibrium conditions for the folding and unfolding of the corresponding system.
  • the benchmark avidin- biotin complex as the receptor-ligand system, which is known to have a particularly strong binding with a dissociation constant KD « 10 15 M.
  • Avidin is a protein composed of four identical subunits, each capable of binding to a single biotin (also known as vitamin H or B7). Therefore, we labelled biotin with a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety to obtain the polarizing ligand (Biotin-TEMPO, Fig. 9F).
  • the saturated protein structure was determined by cryogenic electron microscopy (cryo-EM) and x-ray diffraction (XRD) crystallography (Fig. 9A-E).
  • the two method gave significantly same results.
  • This system is characterized by type of pocket binding site: the ligand is trapped into the protein and its degree of freedom is highly affected.
  • the a 10 A distance between two free radical allows a relative high electron-electron coupling and will affect the O-DNP efficiency, as well.
  • the free radical on the nitroxide group is relatively close (between 4 to 7 A) of polar chemical group such as alcohol or amine group (Water molecules, ARG, LYS, SER, LYS). Few water molecules are present especially on the cry- EM data in the vicinity of nitroxide group.
  • O-DNP effect is very sensitive to the dynamic of water solvent. Moreover, the short distance between two electrons will created a strong electron-electron dipolar coupling that can be affect the relaxation rates and so the O-DNP effect.
  • the 3D structure of the protein/ligand illustrates the complexity for the O-DNP mechanism inside a protein. 18
  • the inset in Fig. 10 shows the 1H-NMR spectra corresponding to a MW irradiation of 8.884 and 8.931 GHz for the free radicals in the reference solution (solid arrow lines) and bound radicals in the complex solution (dashed arrow lines).
  • the general shape is explained by the anisotropy of nitroxide EPR absorbance in fast (free state) and slow (bound state) motion of rotation. In other word, we are able to measure the EPR spectrum with an indirect method without the constraint of having a resonant cavity.
  • the amplitude of O-DNP spectrum gives information in the three main parameters of O-DNP: coupling factor, leakage factor and saturation factor.
  • CRP C-Reactive Protein
  • phosphonooxy- TEMPO 4-Phosphonooxy-2,2,6,6-tetramethyl-1-piperidinyloxyl
  • This system differs from system 1 composed by biotin and avidin.
  • the binding site is at the surface of the protein.
  • the ligand keeps therefore a high degree of freedom and can rotate on its axis.
  • One of the consequences is that the EPR spectrum is less affected by the slow motion mode and the strategy to use two points on the DNP spectrum to create a calibration curve is not pertinent anymore.
  • the distances between two radical are bigger, it means that the contrast between free state and bound state will not be affected by the electron’s coupling effect.
  • the system seems to be simpler because affected by less parameters.
  • the maximal concentration of CRP is 22.4 pM.
  • the consequence of this choice is that the maximal free radical concentration is 112 pM.
  • the calibration curve (Fig. 13) only involves the variating maximum situated at 8.884 GHz, and was constructed from a set of NMR spectra under 8.884 GHz irradiation measured for CRP concentrations between 0 and 24 pM with the same amount of phosphonooxy-TEMPO (120 pM) in each sample. All NMR spectra were measured in high resonance cavity mode with the cavity resonance matching 8.884 GHz. Assuming that the concentration of complex CRP/phosphooxy-TEMPO is proportional of the DNP enhancement, we calculate a KD of 4x1 O 6 M.
  • this versatile method opens the way to the investigation of any system capable of producing a macromolecular complex.
  • the process employs selective 20 molecular probes, it could be applied for the study of dynamic and/or biological systems, potentially for in vivo experiments.
  • it should allow a time-resolved monitoring of complexation, clustering or polymerization processes in situ.
  • the method can be improve by using a high resolution O-DNP setup [21] and considering not only the solvent signal but multiple NMR signal.
  • the method should be appealing as an analytical tool for material science, biochemistry and medicine, potentially providing a new perspective on macromolecular interactions.
  • cryo-EM and XRD proved their utility to characterize the exact position and orientation of free radical. These two methods can be used in the field of DNP, not only O-DNP but also dissolution-DNP and magic angle spinning DNP (MAS-DNP), to create multi-electron systems to optimize DNP mechanism involving specific electron-electron coupling.
  • MAS-DNP magic angle spinning DNP
  • the B magnetic field of 0.295 T was generated with a Varian electromagnet.
  • the microwave irradiation setup was composed of a microwave source Signal Generator Vaunix LMS-123, a 200 Watt traveling wave tube amplifier, a high frequency circulator and a high-power resistance to prevent damage.
  • the EPR resonator was a sapphire tube.
  • the NMR signal was carried out with a home-made setup composed of a pulse generator PulseBlaster Spincore, a PTS 620 frequency synthesizer, a TOMCO RF Pulse Amplifier, a digitizer Gage Applied RazorMax, and a home-made spectrometer.
  • the tuning and matching (T&M) trimmer capacitors (NMTIM120CEK, Municom) were located out of the sample space in an aluminium box.
  • the NMR coil is made in silver with the technic 3D printing and lost wax casting (Materialise NV) and is located into the dielectric resonator.
  • the sample space (EPR resonator and NMR coil) was shielded with a copper cavity.
  • the data were saved in the EER format using the EPU software (TFS) at a magnification of 270kx (pixel size is 0.45 A at the specimen level) and a total dose of 60 electrons per square angstrom (e-/A2) for each exposure, and defocus range of [-0.4- -1.6] micrometers. In total, 14,220 movies were collected.
  • FFS EPU software
  • Image processing 14,220 movies were imported into cryoSparcLive, drift-corrected and 21 dose-weighted, and estimated for contrast transfer function (CTF). After rejection of bad images based on CTF Fit resolution (from 2 to 8A), ice thickness (1-1.2) parameters etc., 8,221 images were used for further processing.
  • Initial picking was performed with Blob Picker, using circular blobs ranging 80-90A of particle diameters, NCC score of 0.267 and Power Score ranging 322-1308. In total, 1341551 particles were picked. Picked particles were extracted with a box size of 384 pixels, and Fourier cropped to 96 pixels (bin 4 times), and subjected to the streaming reference-free 2D classification. The best particles from the best 2D classes were used for the ab-initio structure building and further refined with applying D4 symmetry. After 3D classification the best class was refined to the resolution of 2.1A.
  • Atomic model was re-built manually from the X-ray atomic model with COOTvO.9.6, iterated with rounds of real-space refinement with ligand restraints in PHENIXv1.19.2. After several rounds of refinement side-chains were manually inspected and adjusted for the energy-favored geometry in COOT.

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Abstract

Procédé de détection d'un changement dans une rotation moléculaire d'un système marqué par spin électronique (1) dans une solution. Les spins nucléaires de la solution entière sont mesurés dans la solution à l'aide d'une polarisation dynamique nucléaire Overhauser dans une première mesure de référence, puis les spins nucléaires dudit système marqué par spin électronique (1) et d'au moins une autre molécule (3, 11, 13-16) agrégée et/ou liée à au moins l'un desdits systèmes marqués par spin électronique (1) et non présents dans ladite première mesure, ou desdits systèmes marqués par spin électronique (1) dans des conditions différentes ou dans un environnement différent de celui de la première mesure, sont mesurés dans la solution à l'aide d'une polarisation dynamique nucléaire Overhauser dans une seconde mesure. Une différence dans le spectre d'amélioration de polarisation dynamique nucléaire Overhauser entre lesdites première et seconde mesures est prise en tant que mesure du changement de rotation moléculaire dudit système marqué par spin électronique (1) et/ou de la présence d'un agrégat de ladite au moins une autre molécule (3, 11, 13-16) agrégée et/ou liée à au moins l'un desdits systèmes marqués par spin électronique (1).
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000040988A1 (fr) 1998-12-30 2000-07-13 Nycomed Amersham Plc Essai in vitro recourant a la spectroscopie rmn utilisant l'hyperpolarisation
WO2003091700A2 (fr) 2002-03-26 2003-11-06 Centocor, Inc. Cartographie d'epitopes faisant appel a la resonance magnetique nucleaire
WO2009045551A1 (fr) 2007-10-04 2009-04-09 The General Hospital Corporation Systèmes et procédés à résonance magnétique miniaturisés
US20170029377A1 (en) 2015-07-28 2017-02-02 North Carolina State University Site-specific dynamic nuclear polarization nmr agents

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000040988A1 (fr) 1998-12-30 2000-07-13 Nycomed Amersham Plc Essai in vitro recourant a la spectroscopie rmn utilisant l'hyperpolarisation
WO2003091700A2 (fr) 2002-03-26 2003-11-06 Centocor, Inc. Cartographie d'epitopes faisant appel a la resonance magnetique nucleaire
WO2009045551A1 (fr) 2007-10-04 2009-04-09 The General Hospital Corporation Systèmes et procédés à résonance magnétique miniaturisés
US20170029377A1 (en) 2015-07-28 2017-02-02 North Carolina State University Site-specific dynamic nuclear polarization nmr agents

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BALAKRISHNAN K ET AL: "Monoclonal antibodies to a nitroxide lipid hapten", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 721, no. 1, 13 September 1982 (1982-09-13), pages 30 - 38, XP023473220, ISSN: 0167-4889, [retrieved on 19820913], DOI: 10.1016/0167-4889(82)90020-9 *
BILLER JOSHUA R ET AL: "Perspective of Overhauser dynamic nuclear polarization for the study of soft materials", CURRENT OPINION IN COLLOID & INTERFACE SCIENCE, LONDON, GB, vol. 33, 22 February 2018 (2018-02-22), pages 72 - 85, XP085393551, ISSN: 1359-0294, DOI: 10.1016/J.COCIS.2018.02.007 *
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