Phase-Incremented Steady-State Free Precession as an Alternate Route to High-Resolution NMR.

Pulsed Fourier transform nuclear magnetic resonance (FT-NMR) has reigned supreme in high-resolution, high-field spectroscopy─particularly when targeting complex liquid-state samples involving multiple sharp peaks spread over large spectral bandwidths. It is known, however, that if spectral resolution is not a must, the FT-based approach is not necessarily the optimal route for maximizing NMR sensitivity: if T2 ≈ T1, as often found in solutions, Carr's steady-state free-precession (SSFP) approach can in principle provide a superior signal-to-noise ratio per √(acquisition_time) (SNRt). A rapid train of pulses will then lead to a transverse component that reaches up to 50% of the thermal equilibrium magnetization, provided that pulses are applied at repetition times TR ≪ T2, T1, and that a single suitable offset is involved. It is generally assumed that having to deal with multiple chemical shifts deprives SSFP from its advantages. The present study revisits this assumption by introducing an approach whereby arbitrarily short SSFP-derived free induction decays (FIDs) can deliver high-resolution spectra, without suffering from peak broadenings or phase distortions. To achieve discrimination among nearby frequencies, signals arising from a series of regularly phase-increased excitation pulses are collected. Given SSFP's amplitude and phase sensitivity to the spins' offset, this enables the resolution of sites according to their chemical shift position. In addition, the extreme fold-over associated with SSFP acquisitions is dealt with by a customized discrete FT of the interpulse time-domain signal. Solution-state 13C NMR spectra which compare well with FT-NMR data in terms of sensitivity, bandwidth, and resolution can then be obtained.

Short and long range 2D 15N-15N NMR correlations among peptide groups by novel solid state dipolar mixing schemes.

A recently developed homonuclear dipolar recoupling scheme, Adiabatic Linearly FREquency Swept reCOupling (AL FRESCO), was applied to record two-dimensional (2D) 15N-15N correlations on uniformly 15N-labeled GB1 powders. A major feature exploited in these 15N-15N correlations was AL FRESCO's remarkably low RF power demands, which enabled seconds-long mixing schemes when establishing direct correlations. These 15N-15N mixing schemes proved efficient regardless of the magic-angle spinning (MAS) rate and, being nearly free from dipolar truncation effects, they enabled the detection of long-range, weak dipolar couplings, even in the presence of strong short-range dipolar couplings. This led to a connectivity information that was significantly better than that obtained with spontaneously proton-driven, 15N spin-diffusion experiments. An indirect approach producing long-range 15N-15N correlations was also tested, relying on short (ms-long) 1HN-1HN mixings schemes while applying AL FRESCO chirped pulses along the 15N channel. These indirect mixing schemes produced numerous long-distance Ni-Ni±n (n = 2 - 5) correlations, that might be useful for characterizing three-dimensional arrangements in proteins. Once again, these AL FRESCO mediated experiments proved more informative than variants based on spin-diffusion-based 1HN-1HN counterparts.

Spatiotemporal encoding MRI in a portable low-field system.

PURPOSE: Demonstrate the potential of spatiotemporal encoding (SPEN) MRI to deliver largely undistorted 2D, 3D, and diffusion weighted images on a 110 mT portable system. METHODS: SPEN's quadratic phase modulation was used to subsample the low-bandwidth dimension of echo planar acquisitions, delivering alias-free images with an enhanced immunity to image distortions in a laboratory-built, low-field, portable MRI system lacking multiple receivers. RESULTS: Healthy brain images with different SPEN time-bandwidth products and subsampling factors were collected. These compared favorably to EPI acquisitions including topup corrections. Robust 3D and diffusion weighted SPEN images of diagnostic value were demonstrated, with 2.5 mm isotropic resolutions achieved in 3 min scans. This performance took advantage of the low specific absorption rate and relative long TEs associated with low-field MRI. CONCLUSION: SPEN MRI provides a robust and advantageous fast acquisition approach to obtain faithful 3D images and DWI data in low-cost, portable, low-field systems without parallel acceleration.

Identifying and Overcoming Artifacts in 1 H-Based Saturation Transfer NOE NMR Experiments.

Magnetization transfer experiments are versatile nuclear magnetic resonance (NMR) tools providing site-specific information. We have recently discussed how saturation magnetization transfer (SMT) experiments could leverage repeated repolarizations arising from exchanges between labile and water protons to enhance connectivities revealed via the nuclear Overhauser effect (NOE). Repeated experience with SMT has shown that a number of artifacts may arise in these experiments, which may confound the information being sought - particularly when seeking small NOEs among closely spaced resonances. One of these pertains to what we refer to as "spill-over" effects, originating from the use of long saturation pulses leading to changes in the signals of proximate peaks. A second, related but in fact different effect, derives from what we describe as NOE "oversaturation", a phenomenon whereby the use of overtly intense RF fields overwhelms the cross-relaxation signature. The origin and ways to avoid these two effects are described. A final source of potential artifact arises in applications where the labile 1Hs of interest are bound to 15N-labeled heteronuclei. SMT's long 1H saturation times will then be usually implemented while under 15N decoupling based on cyclic schemes leading to decoupling sidebands. Although these sidebands usually remain invisible in NMR, they may lead to a very efficient saturation of the main resonance when touched by SMT frequencies. All of these phenomena are herein experimentally demonstrated, and solutions to overcome them are proposed.

Relaxation-Assisted Magnetization Transfer Phenomena for a Sensitivity-Enhanced 2D NMR.

2D NOESY and TOCSY play central roles in contemporary NMR. We have recently discussed how solvent-driven exchanges can significantly enhance the sensitivity of such methods when attempting correlations between labile and nonlabile protons. This study explores two scenarios where similar sensitivity enhancements can be achieved in the absence of solvent exchange: the first one involves biomolecular paramagnetic systems, while the other involves small organic molecules in natural abundance. It is shown that, in both cases, the effects introduced by either differential paramagnetic shift and relaxation or by polarization sharing among networks of protons can provide a similar sensitivity boost, as previously discussed for solvent exchange. The origin and potential of the resulting enhancements are analyzed, and experiments that demonstrate them in protein and natural products are exemplified. Limitations and future improvements of these approaches are also briefly discussed.

Prostate lesions characterization using diffusion-weighted spatiotemporal encoded MRI: Feasibility and initial assessment.

PURPOSE: To assess the feasibility and reliability of a DWI protocol based on spatiotemporally encoding (SPEN), to target prostate lesions along guidelines normally used in EPI-based DWI clinical practice. METHODS: Prostate Imaging-Reporting and Data System recommendations underlying clinical prostate scans were used to develop a SPEN-based DWI protocol, which included a novel, local, low-rank regularization algorithm. These DWI acquisitions were run at 3 T under similar nominal spatial resolutions and diffusion-weighting b-values as used in EPI-based clinical studies. Prostates of 11 patients suspected of clinically significant prostate cancer lesions were therefore scanned using the two methods, with the same number of slices, same slice thickness, and same interslice gaps. RESULTS: Of the 11 patients scanned, SPEN and EPI provided comparable information in 7 of the cases, whereas EPI was deemed superior in a case for which SPEN images had to be acquired with a shorter effective TR owing to scan-time constraints. SPEN provided reduced susceptibility to field-derived distortions in 3 of the cases. CONCLUSIONS: SPEN's ability to provide prostate lesion contrast was most clearly evidenced for DW images acquired with b ≥ 900 s/mm2 . SPEN also succeeded in decreasing occasional image distortions in regions close to the rectum, affected by field inhomogeneities. EPI advantages arose when using short effective TRs, a regime in which SPEN-based DWI was handicapped by its use of nonselective spin inversions, leading to the onset of an additional T1 weighting.

Understanding aliasing effects and their removal in SPEN MRI: A k-space perspective.

PURPOSE: To characterize the mechanism of formation and the removal of aliasing artifacts and edge ghosts in spatiotemporally encoded (SPEN) MRI within a k-space theoretical framework. METHODS: SPEN's quadratic phase modulation can be described in k-space by a convolution matrix whose coefficients derive from Fourier relations. This k-space model allows us to pose SPEN's reconstruction as a deconvolution process from which aliasing and edge ghost artifacts can be quantified by estimating the difference between a full sampling and reconstructions resulting from undersampled SPEN data. RESULTS: Aliasing artifacts in SPEN MRI reconstructions can be traced to image contributions corresponding to high-frequency k-space signals. The k-space picture provides the spatial displacements, phase offsets, and linear amplitude modulations associated to these artifacts, as well as routes to removing these from the reconstruction results. These new ways to estimate the artifact priors were applied to reduce SPEN reconstruction artifacts on simulated, phantom, and human brain MRI data. CONCLUSION: A k-space description of SPEN's reconstruction helps to better understand the signal characteristics of this MRI technique, and to improve the quality of its resulting images.

High-resolution multi-shot diffusion-weighted MRI combining markerless prospective motion correction and locally low-rank constrained reconstruction.

PURPOSE: Subject head motion is a major challenge in DWI, leading to image blurring, signal losses, and biases in the estimated diffusion parameters. Here, we investigate a combined application of prospective motion correction and spatial-angular locally low-rank constrained reconstruction to obtain robust, multi-shot, high-resolution diffusion-weighted MRI under substantial motion. METHODS: Single-shot EPI with retrospective motion correction can mitigate motion artifacts and resolve any mismatching of gradient encoding orientations; however, it is limited by low spatial resolution and image distortions. Multi-shot acquisition strategies could achieve higher resolution and image fidelity but increase the vulnerability to motion artifacts and phase variations related to cardiac pulsations from shot to shot. We use prospective motion correction with optical markerless motion tracking to remove artifacts and reduce image blurring due to bulk motion, combined with locally low-rank regularization to correct for remaining artifacts due to shot-to-shot phase variations. RESULTS: The approach was evaluated on healthy adult volunteers at 3 Tesla under different motion patterns. In multi-shot DWI, image blurring due to motion with 20 mm translations and 30° rotations was successfully removed by prospective motion correction, and aliasing artifacts caused by shot-to-shot phase variations were addressed by locally low-rank regularization. The ability of prospective motion correction to preserve the orientational information in DTI without requiring a reorientation of the b-matrix is highlighted. CONCLUSION: The described technique is proved to hold valuable potential for mapping brain diffusivity and connectivity at high resolution for studies in subjects/cohorts where motion is common, including neonates, pediatrics, and patients with neurological disorders.

High-field ex vivo and in vivo two-dimensional nuclear magnetic resonance spectroscopy in murine brain: Resolving and exploring the molecular environment.

The structural and chemical complexities within the brain pose a challenge that few noninvasive techniques can tackle with the dexterity of nuclear magnetic resonance (NMR) spectroscopy. Still, even with the advent of ultrahigh fields and of cryogenically cooled coils for in vivo research, the superposition of metabolic resonances arising from the brain remains a challenge. The present study explores the potential to tackle this milieu using a combination of two-dimensional (2D) NMR techniques, implemented on murine brains in vivo at 15.2 T and ex vivo at 14.1 T. While both experiments were affected by substantial inhomogeneous broadenings conveying distinct elongated lineshapes to the cross-peaks, the ability of increased fields to resolve off-diagonal resonances was clear. A comparison between the corresponding conventional and double quantum-filtered correlated spectroscopy traces enabled an improved assignment of in vivo resonances on the basis of more sensitive ex vivo 2D acquisitions, foremost on the basis of homonuclear cross-relaxation-driven correlations for peaks resonating downfield from water, and of heteronuclear correlations at natural abundance for the upfield protons. With the aid of such 2D correlations approximately 29 metabolites could be resolved and identified. This enhanced resolution was used to explore features related to the metabolites' diffusivities, their exposure to water, and their facility to undergo magnetization transfers to amide/amine/hydroxyl resonances. Cross-peaks from main murine brain biomolecules, including choline, creatine, γ-aminobutyric acid, N-acetyl aspartate, glutamine, and glutamate, showed enhancements in several of these various features, opening interesting vistas about metabolite compartmentalization as viewed by these 2D NMR experiments.

Deuterium imaging of the Warburg effect at sub-millimolar concentrations by joint processing of the kinetic and spectral dimensions.

Deuterium metabolic imaging (DMI) is a promising molecular MRI approach, which follows the administration of deuterated substrates and their metabolization. [6,6'-2 H2 ]-glucose for instance is preferentially converted in tumors to [3,3'-2 H2 ]-lactate as a result of the Warburg effect, providing a distinct resonance whose mapping using time-resolved spectroscopic imaging can diagnose cancer. The MR detection of low-concentration metabolites such as lactate, however, is challenging. It has been recently shown that multi-echo balanced steady-state free precession (ME-bSSFP) increases the signal-to-noise ratio (SNR) of these experiments approximately threefold over regular chemical shift imaging; the present study examines how DMI's sensitivity can be increased further by advanced processing methods. Some of these, such as compressed sensing multiplicative denoising and block-matching/3D filtering, can be applied to any spectroscopic/imaging methods. Sensitivity-enhancing approaches were also specifically tailored to ME-bSSFP DMI, by relying on priors related to the resonances' positions and to features of the metabolic kinetics. Two new methods are thus proposed that use these constraints for enhancing the sensitivity of both the spectral images and the metabolic kinetics. The ability of these methods to improve DMI is evidenced in pancreatic cancer studies carried at 15.2 T, where suitable implementations of the proposals imparted eightfold or more SNR improvement over the original ME-bSSFP data, at no informational cost. Comparisons with other propositions in the literature are briefly discussed.

Cross polarization from dipolar-order under magic angle spinning: The ADRF-CPMAS NMR experiment.

Techniques for enhancing the signals arising from low-γ, insensitive (I) nuclei are central to solid-state nuclear magnetic resonance. One of the leading and best-established methods to sensitize these unreceptive species is Hartmann-Hahn cross polarization (HH-CP), a polarization transfer mechanism often executed under MAS. Herein, we explore the possibility of utilizing the 1H dipolar order created via adiabatic demagnetization in the rotating frame (ADRF), to enhance the unreceptive spins under MAS. It is found that an efficient polarization transfer via ADRF-CPMAS is not only possible but can exceed, at least in some instances involving plastic crystals, the efficiency of an optimized HH-CPMAS transfer. The experiment requires low radiofrequency nutation fields on both the 1H- and the I-spin channels, and displays unusual matching conditions that are reminiscent of the zero- and double-quantum matching conditions arising under CPMAS, albeit centered at zero frequency and demanding the simultaneous involvement of several spins. The origin of these multi-spin transfer processes is analytically derived and numerically simulated in predictions that compare well with experimental 13C and 15N results collected on model compounds at different spinning speeds. These derivations start from descriptions that depart from traditional thermodynamic arguments, and treat instead the ADRF processes in static and spinning solids on the basis of coherent evolutions. The predictions of these analytical derivations are corroborated by numerical simulations. The effects of additional factors, including chemical shift anisotropies, J-couplings, and radiofrequency inhomogeneities, are also theoretically and experimentally explored.

Indirectly detected satellite-transition quadrupolar NMR via progressive saturation of the proton reservoir.

Static satellite-transitions (ST) NMR line shapes from half-integer quadrupolar nuclei could be very informative: they can deliver insight about local motions over a wide range of timescales, and can report on small changes in the local electronic environments as reflected by variations in the quadrupolar parameters. Satellite transitions, however, are typically "invisible" for half-integer quadrupolar nuclei due to their sheer breadth, leading to low signal-to-noise ratio -especially for unreceptive low-gamma or dilute quadrupolar nuclei. Very recently we have introduced a method for enhancing the NMR sensitivity of unreceptive X nuclei in static solids dubbed PROgressive Saturation of the Proton Reservoir (PROSPR), which opens the possibility of magnifying the signals from such spins by repeatedly imprinting frequency-selective X-driven depolarizations on the much more sensitive 1H NMR signal. Here, we show that PROSPR's efficacy is high enough for enabling the detection of static ST NMR for challenging species like 35Cl, 33S and even 17O -all at natural-abundance. The ensuing ST-PROSPR NMR experiment thus opens new approaches to probe ultra-wideline (6-8 MHz wide) spectra. These highly pronounced anisotropies can in turn deliver new vistas about dynamic changes in solids, as here illustrated by tracking ST line shapes as a function of temperature during thermally-driven events.

A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR.

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW's enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-1H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.

Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR.

INEPT-based experiments are widely used for 1 H→15 N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the Hwater ↔ ${ \leftrightarrow }$ HN exchange process to boost the 1 H→15 N transfer process. This leveraging, however, demands the simultaneous spin-locking of both Hwater and HN protons by a strong 1 H RF field, while fulfilling the γH B1,H =γN B1,N Hartmann-Hahn matching condition. Given the low value of γN /γH , however, these demands are often incompatible-particularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-à-vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.

Harnessing Water to Enhance Quadrupolar NMR Spectroscopy and Imaging.

17 O and 14 N are attractive targets for in vivo NMR spectroscopy and imaging, but low gyromagnetic ratios γ and fast spin relaxation complicate observations. This work explores indirect ways of detecting some of these sites with the help of proton-detected double resonance techniques. As standard coherence transfer methods are of limited use for such indirect detection, alternative routes for probing the quadrupolar spectra on 1 H were tested. These centered on modulating the broadening effects imparted onto protons adjacent to the low-γ species through J couplings through either continuous wave or spin-echo double-resonance decoupling/recoupling sequences. As in all cases, the changes imparted by these double-resonance strategies were small due to the fast relaxation undergone by the quadrupoles, the sensitivity of these approaches was amplified by transferring their effects onto the abundant water 1 H signal. These amplifications were mediated by the spontaneous exchanges that the labile 1 Hs bound to 17 O or 14 N undergo with the water protons. In experiments designed on the basis of double-resonance spin echoes, these enhancements were imparted by looping the transverse encodings together with multiple longitudinal storage periods, leading to decoupling-recoupling with exchange (D-REX) sequences. In experiments designed on the basis of continuous on/off quadrupolar decoupling, these solvent exchanges were incorporated into chemical-exchange saturation transfer schemes, leading to decoupling-recoupling with saturation transfer (D-REST) sequences. Both of these variants harnessed sizable proportions of the easily detectable water signals, in order to characterize the NMR spectra and/or to image with atomic-site specificity the 17 O and 14 N species.

The Extended Hadamard Transform: Sensitivity-Enhanced NMR Experiments Among Labile and Non-Labile 1 Hs of SARS-CoV-2-derived RNAs.

Hadamard encoded saturation transfer can significantly improve the efficiency of NOE-based NMR correlations from labile protons in proteins, glycans and RNAs, increasing the sensitivity of cross-peaks by an order of magnitude and shortening experimental times by ≥100-fold. These schemes, however, fail when tackling correlations within a pool of labile protons - for instance imino-imino correlations in RNAs or amide-amide correlations in proteins. Here we analyze the origin of the artifacts appearing in these experiments and propose a way to obtain artifact-free correlations both within the labile pool as well as between labile and non-labile 1 Hs, while still enjoying the gains arising from Hadamard encoding and solvent repolarizations. The principles required for implementing what we define as the extended Hadamard scheme are derived, and its clean, artifact-free, sensitivity-enhancing performance is demonstrated on RNA fragments derived from the SARS-CoV-2 genome. Sensitivity gains per unit time approaching an order of magnitude are then achieved in both imino-imino and imino-amino/aromatic protons 2D correlations; similar artifact-free sensitivity gains can be observed when carrying out extended Hadamard encodings of 3D NOESY/HSQC-type experiments. The resulting spectra reveal significantly more correlations than their conventionally acquired counterparts, which can support the spectral assignment and secondary structure determination of structured RNA elements.

J-Driven dynamic nuclear polarization for sensitizing high field solution state NMR.

Dynamic nuclear polarization (DNP) is widely used to enhance solid state nuclear magnetic resonance (NMR) sensitivity. Its efficiency as a generic signal-enhancing approach for liquid state NMR, however, decays rapidly with magnetic field B0, unless mediated by scalar interactions arising only in exceptional cases. This has prevented a more widespread use of DNP in structural and dynamical solution NMR analyses. This study introduces a potential solution to this problem, relying on biradicals with exchange couplings Jex of the order of the electron Larmor frequency ωE. Numerical and analytical calculations show that in such Jex ≈ ±ωE cases a phenomenon akin to that occurring in chemically induced DNP (CIDNP) happens, leading to different relaxation rates for the biradical singlet and triplet states which are hyperfine-coupled to the nuclear α or ß states. Microwave irradiation can then generate a transient nuclear polarization build-up with high efficiency, at all magnetic fields that are relevant in contemporary NMR, and for all rotational diffusion correlation times that occur in small- and medium-sized molecules in conventional solvents.

3D relaxation-assisted separation of wideline solid-state NMR patterns for achieving site resolution.

There are currently no methods for the acquisition of ultra-wideline (UW) solid-state NMR spectra under static conditions that enable reliable separation and resolution of overlapping powder patterns arising from magnetically distinct nuclei. This stands in contrast to the variety of techniques available for spin-1/2 or half-integer quadrupolar nuclei with narrow central transition patterns under magic-angle spinning (MAS). Resolution of overlapping signals is routinely achieved in MRI and solution-state NMR by exploiting relaxation differences between nonequivalent sites. Preliminary studies of relaxation assisted separation (RAS) for separating overlapping UWNMR patterns using pseudo-inverse Laplace Transforms have reported two-dimensional spectra featuring relaxation rates correlated to NMR interaction frequencies. However, RAS methods are inherently sensitive to experimental noise, and require that relaxation rates associated with overlapped patterns be significantly different from one another. Herein, principal component analysis (PCA) denoising is implemented to increase the signal-to-noise ratios of the relaxation datasets and RAS routines are stabilized with truncated singular value decomposition (TSVD) and elastic net (EN) regularization to resolve overlapped patterns with a larger tolerance for differences in relaxation rates. We extend these methods for improved pattern resolution by utilizing 3D frequency-R1-R2 correlation spectra. Synthetic and experimental datasets, including 35Cl (I = 3/2), 2H (I = 1), and 14N (I = 1) NMR of organic and biological compounds, are explored with both regularized 2D RAS and 3D RAS; comparison of these data reveal improved resolution in the latter case. These methods have great potential for separating overlapping powder patterns under both static and MAS conditions.

On the potential of Fourier-encoded saturation transfers for sensitizing solid-state magic-angle spinning NMR experiments.

Chemical exchange saturation transfer (CEST) is widely used for enhancing the solution nuclear magnetic resonance (NMR) signatures of magnetically dilute spin pools, in particular, species at low concentrations undergoing chemical exchanges with an abundant spin pool. CEST's main feature involves encoding and then detecting weak NMR signals of the magnetically dilute spin pools on a magnetically abundant spin pool of much easier detection, for instance, the protons of H2O. Inspired by this method, we propose and exemplify a methodology to enhance the sensitivity of magic-angle spinning (MAS) solid-state NMR spectra. Our proposal uses the abundant 1H reservoir arising in organic solids as the magnetically abundant spin pool and relies on proton spin diffusion in lieu of chemical exchange to mediate polarization transfer between a magnetically dilute spin pool and this magnetically abundant spin reporter. As an initial test of this idea, we target the spectroscopy of naturally abundant 13C and rely on a Fourier-encoded version of the CEST experiment for achieving broadbandness in coordination with both MAS and heteronuclear decoupling, features normally absent in CEST. Arbitrary evolutions of multiple 13C sites can, thus, be imprinted on the entire 1H reservoir, which is subsequently detected. Theoretical predictions suggest that orders-of-magnitude signal enhancements should be achievable in this manner, on the order of the ratio between the 13C and the 1H reservoirs' abundances. Experiments carried out under magic-angle spinning conditions evidenced 5-10× gains in signal amplitudes. Further opportunities and challenges arising in this Fourier-encoded saturation transfer MAS NMR approach are briefly discussed.

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. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.