H-F cross-polarization magic angle spinning dynamic nuclear polarization NMR investigation of advanced pharmaceutical formulations.

A new 3.2 mm H-F-X magic angle spinning dynamic nuclear polarization NMR (MAS DNP-NMR) probe was developed with a unique coil design with separate radiofrequency channels for H excitation and C or F detection to enable acquisition of H-F cross-polarization (CP) MAS experiments, direct-detected F spectra with proton decoupling, and acquisition on C with simultaneous double decoupling on the H and 19F channels as well as H-F-C double-CP experiments under low temperature MAS DNP conditions. We use these sequences to study AZD2811, which is an active pharmaceutical ingredient (API), in its pure dry state as well as in its corresponding drug delivery formulation consisting of drug-loaded polymeric nanoparticles (PNPs).

Polarizing agents for efficient high field DNP solid-state NMR spectroscopy under magic-angle spinning: from design principles to formulation strategies.

Dynamic Nuclear Polarization (DNP) has recently emerged as a cornerstone approach to enhance the sensitivity of solid-state NMR spectroscopy under Magic Angle Spinning (MAS), opening unprecedented analytical opportunities in chemistry and biology. DNP relies on a polarization transfer from unpaired electrons (present in endogenous or exogenous polarizing agents) to nearby nuclei. Developing and designing new polarizing sources for DNP solid-state NMR spectroscopy is currently an extremely active research field , that has recently led to significant breakthroughs and key achievements, in particular at high magnetic fields.

Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR.

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation.

Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy.

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R relaxation dispersion NMR spectroscopy (RD) determines chemical shift differences between the most stable, ground state and the short-lived, low-populated excited states. We describe a procedure for deducing the excited state structure from these chemical shift differences using the mutate-and-chemical-shift-fingerprint (MCSF) method, which requires ~2-6 weeks and moderate understanding of NMR and RNA structure.

Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA.

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1ρ relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.

H, C and N chemical shift assignment of the stem-loop 5a from the 5'-UTR of SARS-CoV-2.

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets.

Nuclear spin noise tomography in three dimensions with iterative simultaneous algebraic reconstruction technique (SART) processing.

We report three-dimensional spin noise imaging (SNI) of nuclear spin density from spin noise data acquired by Faraday detection. Our approach substantially extends and improves the two-dimensional SNI method for excitation-less magnetic resonance tomography reported earlier (Müller and Jerschow, 2006). This proof of principle was achieved by taking advantage of the particular continuous nature of spin noise acquired in the presence of constant magnitude magnetic field gradients and recent advances in nuclear spin noise spectroscopy acquisition as well as novel processing techniques.

Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA. Here we use R relaxation-dispersion nuclear magnetic resonance and molecular simulations to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed.

Observing an Antisense Drug Complex in Intact Human Cells by in-Cell NMR Spectroscopy.

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy.

RNA Dynamics by NMR Spectroscopy.

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution.

Refocused linewidths less than 10 Hz in H solid-state NMR.

Coherence lifetimes in homonuclear dipolar decoupled H solid-state NMR experiments are usually on the order of a few ms. We discover an oscillation that limits the lifetime of the coherences by recording spin-echo dephasing curves. We find that this oscillation can be removed by the application of a double spin-echo experiment, leading to coherence lifetimes of more than 45 ms in adamantane and more that 22 ms in β-AspAla, corresponding to refocused linewidths of less than 7 and 14 Hz respectively.

Efficient Detection of Structure and Dynamics in Unlabeled RNAs: The SELOPE Approach.

The knowledge of structure and dynamics is crucial to explain the function of RNAs. While nuclear magnetic resonance (NMR) is well suited to probe these for complex biomolecules, it requires expensive, isotopically labeled samples, and long measurement times. Here we present SELOPE, a new robust, proton-only NMR method that allows us to obtain site-specific overview of structure and dynamics in an entire RNA molecule using an unlabeled sample.

Comprehensive analysis of NMR data using advanced line shape fitting.

NMR spectroscopy is uniquely suited for atomic resolution studies of biomolecules such as proteins, nucleic acids and metabolites, since detailed information on structure and dynamics are encoded in positions and line shapes of peaks in NMR spectra. Unfortunately, accurate determination of these parameters is often complicated and time consuming, in part due to the need for different software at the various analysis steps and for validating the results. Here, we present an integrated, cross-platform and open-source software that is significantly more versatile than the typical line shape fitting application.

Nonlinear detection of secondary isotopic chemical shifts in NMR through spin noise.

The detection of minor species in the presence of large amounts of similar main components remains a key challenge in analytical chemistry, for instance, to obtain isotopic fingerprints. As an alternative to the classical NMR scheme based on coherent excitation and detection, here we introduce an approach based on spin-noise detection. Chemical shifts and transverse relaxation rates are determined using only the detection circuit.

Capturing Excited States in the Fast-Intermediate Exchange Limit in Biological Systems Using H NMR Spectroscopy.

Changes in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high-energy states.

A solid-state NMR method to determine domain sizes in multi-component polymer formulations.

Polymer domain sizes are related to many of the physical properties of polymers. Here we present a solid-state NMR experiment that is capable of measuring domain sizes in multi-component mixtures. The method combines selective excitation of carbon magnetization to isolate a specific component with proton spin diffusion to report on domain size.

Nanostructure of Materials Determined by Relayed Paramagnetic Relaxation Enhancement.

Particle and domain sizes strongly influence the properties of materials. Here we present an NMR approach based on paramagnetic relaxation enhancement (PRE) relayed by spin diffusion (SD), which allows us to determine lengths in the nm-μm range. We demonstrate the method on multicomponent organic polymer mixtures by selectively doping one component with a paramagnetic center in order to measure the domain size in a second component.

High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy.

We demonstrate that high field (9.4 T) dynamic nuclear polarization (DNP) at cryogenic (∼100 K) sample temperatures enables the rapid acquisition of natural abundance (1)H-(2)H cross-polarization magic angle spinning (CPMAS) solid-state NMR spectra of organic solids. Spectra were obtained by impregnating substrates with a solution of the stable DNP polarizing agent TEKPol in tetrachloroethane.

Resonance assignment of PsbP: an extrinsic protein from photosystem II of Spinacia oleracea.

PsbP (23 kDa) is an extrinsic eukaryotic protein of photosystem II found in the thylakoid membrane of higher plants and green algae. It has been proven to be indispensable for proper functioning of the oxygen evolving complex. By interaction with other extrinsic proteins (PsbQ, PsbO and PsbR), it modulates the concentration of two cofactors of the water splitting reaction, Ca(2+) and Cl(-).

On the tuning of high-resolution NMR probes.

Three optimum conditions for the tuning of NMR probes are compared: the conventional tuning optimum, which is based on radio-frequency pulse efficiency, the spin noise tuning optimum based on the line shape of the spin noise signal, and the newly introduced frequency shift tuning optimum, which minimizes the frequency pushing effect on strong signals. The latter results if the radiation damping feedback field is not in perfect quadrature to the precessing magnetization. According to the conventional RLC (resistor-inductor-capacitor) resonant circuit model, the optima should be identical, but significant deviations are found experimentally at low temperatures, in particular on cryogenically cooled probes.

The first observation of Carbon-13 spin noise spectra.

We demonstrate the first (13)C NMR spin noise spectra obtained without any pulse excitation by direct detection of the randomly fluctuating noise from samples in a cryogenically cooled probe. Noise power spectra were obtained from (13)C enriched methanol and glycerol samples at 176 MHz without and with (1)H decoupling, which increases the sensitivity without introducing radio frequency interference with the weak spin noise. The multiplet amplitude ratios in (1)H coupled spectra indicate that, although pure spin noise prevails in these spectra, the influence of absorbed circuit noise is still significant at the high concentrations used.