Multiplexing experiments in NMR and multi-nuclear MRI

Multiplexing NMR experiments by direct detection of multiple free induction decays (FIDs) in a single experiment offers a dramatic increase in the spectral information content and often yields significant improvement in sensitivity per unit time. Experiments with multi-FID detection have been designed with both homonuclear and multinuclear acquisition, and the advent of multiple receivers on commercial spectrometers opens up new possibilities for recording spectra from different nuclear species in parallel. Here we provide an extensive overview of such techniques, designed for applications in liquid-and solidstate NMR as well as in hyperpolarized samples. A brief overview of multinuclear MRI is also provided, to stimulate cross fertilization of ideas between the two areas of research (NMR and MRI). It is shown how such techniques enable the design of experiments that allow structure elucidation of small molecules from a single measurement. Likewise, in biomolecular NMR experiments multi-FID detection allows complete resonance assignment in proteins. Probes with multiple RF microcoils routed to multiple NMR receivers provide an alternative way of increasing the throughput of modern NMR systems, effectively reducing the cost of NMR analysis and increasing the information content at the same time. Solidstate NMR experiments have also benefited immensely from both parallel and sequential multi-FID detection in a variety of multi-dimensional pulse schemes. We are confident that multi-FID detection will become an essential component of future NMR methodologies, effectively increasing the sensitivity and information content of NMR measurements. (c) 2021 Elsevier B.V. All rights reserved

Broadband 180 degree universal rotation pulses for NMR spectroscopy designed by optimal control

Broadband inversion pulses that rotate all magnetization components 180 degrees about a given fixed axis are necessary for refocusing and mixing in high-resolution NMR spectroscopy. The relative merits of various methodologies for generating pulses suitable for broadband refocusing are considered. The de novo design of 180 degree universal rotation pulses using optimal control can provide improved performance compared to schemes which construct refocusing pulses as composites of existing pulses. The advantages of broadband universal rotation by optimized pulses (BURBOP) are most evident for pulse design that includes tolerance to RF inhomogeneity or miscalibration. We present new modifications of the optimal control algorithm that incorporate symmetry principles and relax conservative limits on peak RF pulse amplitude for short time periods that pose no threat to the probe. We apply them to generate a set of pulses suitable for widespread use in Carbon-13 spectroscopy on the majority of available probes

A non-invasive subtle pulse rate extraction method based on Eulerian video magnification

Measuring pulse rate by means of video recording of the wrist area is a non-invasive approach. Eulerian Video Linear Magnification (EVLM) is used in this paper to magnify, and make visible, subtle pulse-induced wrist motions. A series of experiments are conducted to investigate the performance of ELVM under various conditions, such as light intensity, background colour and a set of video recording parameters. The results show that a light intensity of around 224 to 229 lx is optimal; excess or inadequate light significantly impairs the success of amplifying the skin movement resulting from the pulse. It is demonstrated that a white background colour enables both the radial and ulnar areas to be clearly visible in the recorded video, thus improving pulse measurement. In addition, it is shown that a female’s pulse strength is approximately 40 % weaker than that of a male averaged over the participants

Mathematical treatment of adiabatic fast passage pulses for the computation of nuclear spin relaxation rates in proteins with conformational exchange

Although originally designed for broadband inversion and decoupling in NMR spectroscopy, recent methodological developments have introduced adiabatic fast passage (AFP) pulses into the field of protein dynamics. AFP pulses employ a frequency sweep, and have not only superior inversion properties with respect to offset effects, but they are also easily implemented into a pulse sequence. As magnetization is dragged from the +z to the −z direction, Larmor precession is impeded since magnetization becomes spin-locked, which is a potentially useful feature for the investigation of microsecond to millisecond dynamics. A major drawback of these pulses as theoretical prediction is concerned, however, results from their time-dependent offset: simulations of spin density matrices under the influence of a time-dependent Hamiltonian with non-commuting elements are costly in terms of computational time, rendering data analysis impracticable. In this paper we suggest several ways to reduce the computational time without compromising accuracy with respect to effects such as cross-correlated relaxation and modulation of the chemical shift

Selective 13C labeling of nucleotides for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle

Escherichia coli (E. coli) is an ideal organism to tailor-make labeled nucleotides for biophysical studies of RNA. Recently, we showed that adding labeled formate enhanced the isotopic enrichment at protonated carbon sites in nucleotides. In this paper, we show that growth of a mutant E. coli strain DL323 (lacking succinate and malate dehydrogenases) on 13C-2-glycerol and 13C-1,3-glycerol enables selective labeling at many useful sites for RNA NMR spectroscopy. For DL323 E. coli grown in 13C-2-glycerol without labeled formate, all the ribose carbon atoms are labeled except the C3′ and C5′ carbon positions. Consequently the C1′, C2′ and C4′ positions remain singlet. In addition, only the pyrimidine base C6 atoms are substantially labeled to ~96% whereas the C2 and C8 atoms of purine are labeled to ~5%. Supplementing the growth media with 13C-formate increases the labeling at C8 to ~88%, but not C2. Not unexpectedly, addition of exogenous formate is unnecessary for attaining the high enrichment levels of ~88% for the C2 and C8 purine positions in a 13C-1,3-glycerol based growth. Furthermore, the ribose ring is labeled in all but the C4′ carbon position, such that the C2′ and C3′ positions suffer from multiplet splitting but the C5′ position remains singlet and the C1′ position shows a small amount of residual C1′–C2′ coupling. As expected, all the protonated base atoms, except C6, are labeled to ~90%. In addition, labeling with 13C-1,3-glycerol affords an isolated methylene ribose with high enrichment at the C5′ position (~90%) that makes it particularly attractive for NMR applications involving CH2-TROSY modules without the need for decoupling the C4′ carbon. To simulate the tumbling of large RNA molecules, perdeuterated glycerol was added to a mixture of the four nucleotides, and the methylene TROSY experiment recorded at various temperatures. Even under conditions of slow tumbling, all the expected carbon correlations were observed, which indicates this approach of using nucleotides obtained from DL323 E. coli will be applicable to high molecular weight RNA systems

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR

Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1′ and C5′ with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg2+ ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly 13C/15N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive 13C–13C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules

Site-specific labeling of nucleotides for making RNA for high resolution NMR studies using an E. coli strain disabled in the oxidative pentose phosphate pathway

Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and C6 positions of the pyrimidine rings are labeled with minimal multiplet splitting due to 1JC5C6 scalar coupling. However, the C2/C8 sites within purine rings and the C1′/C3′/C5′ positions within the ribose rings have reduced labeling. Addition of 13C-labeled formate leads to increased labeling at the base C2/C8 and the ribose C1′/C3′/C5′ positions; these new specific labels result in two- to three-fold increase in the number of resolved resonances. This use of formate and 15N-ammonium sulfate promises to extend further the utility of these alternate site specific labels to make labeled RNA for downstream biophysical applications such as structural, dynamics and functional studies of interesting biologically relevant RNAs

Distinguishing closely related amyloid precursors using an RNA aptamer

Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ?2-microglobulin (h?2m) can co-polymerize with its N-terminally truncated variant (?N6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using h?2m and its amyloidogenic counterpart, ??6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds h?2m with an EC50 of ?200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-h?2m complex, revealing that the aptamer binds to the face of h?2m containing the A, B, E, and D ?-strands. In contrast, binding of B6 to ?N6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of h?2m and ?N6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity