Constraints on the structure of (CUG)97 RNA from magic-angle-spinning solid-state NMR spectroscopy.
Explore the source record for details and available documents.
3' Untranslated Regions↗
Biomedical subjects
Jörg Leppert
Publications and source records attributed to Jörg Leppert.
Tailoring broadband inversion pulses for MAS solid state NMR.
A simple approach is demonstrated for designing optimised broadband inversion pulses for MAS solid state NMR studies of biological systems. The method involves a two step numerical optimisation procedure and takes into account experimental requirements such as the pulse length, resonance offset range and extent of H(1) inhomogeneity compensation needed. A simulated annealing protocol is used initially to find appropriate values for the parameters that define the well known tanh/tan adiabatic pulse such that a satisfactory spin inversion is achieved with minimum RF field strength. This information is then used in the subsequent stage of refinement where the RF pulse characteristics are further tailored via a local optimisation procedure without imposing any restrictions on the amplitude and frequency modulation profiles. We demonstrate that this approach constitutes a generally applicable tool for obtaining pulses with good inversion characteristics. At moderate MAS frequencies the efficacy of the method is experimentally demonstrated for generating double-quantum NMR spectra via the zero-quantum dipolar recoupling scheme RFDR.
Carbon Isotopes↗
Solid state NMR at high magic angle spinning frequencies: dipolar chemical shift correlation with adiabatic inversion pulse based RF pulse schemes.
The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H(1) inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated.
Carbon Isotopes↗
TEDOR with adiabatic inversion pulses: Resonance assignments of 13C/15N labelled RNAs.
We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.
Carbon Isotopes↗
Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy.
It is demonstrated that the spatial proximity of (1)H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)(97) that has been implicated in the neuromuscular disease myotonic dystrophy. (15)N-(15)N chemical shift correlation studies confirm the presence of Watson-Crick GC base pairs in (CUG)(97).
Base Pairing↗
Identification of NH...N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy.
RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH...N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6-2.9 A. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH.N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.
Base Pairing↗
(1)H, (13)C and (15)N sequence-specific resonance assignments of the two-domain thrombin inhibitor dipetalin.
Explore the source record for details and available documents.
Animals↗
Homonuclear chemical shift correlation in rotating solids via RNnu n symmetry-based adiabatic RF pulse schemes.
The efficacy of RN(nu) (n) symmetry-based adiabatic Zero-Quantum (ZQ) dipolar recoupling schemes for obtaining chemical shift correlation data at moderate magic angle spinning frequencies has been evaluated. RN(nu) (n) sequences generally employ basic inversion elements that correspond to a net 180 degrees rotation about the rotating frame x-axis. It is shown here via numerical simulations and experimental measurements that it is also possible to achieve efficient ZQ dipolar recoupling via RN(nu) (n) schemes employing adiabatic pulses. Such an approach was successfully used for obtaining (13)C chemical shift correlation spectra of a uniformly labelled sample of (CUG)(97) - a triplet repeat expansion RNA that has been implicated in the neuromuscular disease myotonic dystrophy. An analysis of the (13)C sugar carbon chemical shifts suggests, in agreement with our recent (15)N MAS-NMR studies, that this RNA adopts an Alpha-helical conformation.
Nuclear Magnetic Resonance, Biomolecular↗
RFDR with adiabatic inversion pulses: application to internuclear distance measurements.
In the context of the structural characterisation of biomolecular systems via MAS solid state NMR, the potential utility of homonuclear dipolar recoupling with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. The results obtained suggest that it is possible to obtain reliable estimates of internuclear distances via an analysis of the initial cross-peak intensity buildup curves generated from two-dimensional adiabatic inversion pulse driven longitudinal magnetisation exchange experiments.
Computer Simulation↗
Triple resonance MAS NMR with (13C, 15N) labelled molecules: reduced dimensionality data acquisition via 13C-15N heteronuclear two-spin coherence transfer pathways.
A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly ((13)C, (15)N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear (13)C-(15)N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power (1)H decoupling in experiments requiring long t (1) acquisition times.
Carbon Isotopes↗
Adiabatic TOBSY in rotating solids.
A MAS solid state NMR approach for achieving efficient scalar coupling mediated through-bond (13)C chemical shift correlations of the aliphatic carbons in uniformly labelled peptides/proteins is described. The method involves the application of a continuous train of adiabatic inversion pulses, as in the adiabatic TOCSY experiments carried out in solution state NMR studies. While rotor synchronised application of adiabatic inversion pulses leads to dipolar correlations, it is shown here via numerical simulations and experimental measurements that asynchronous application of adiabatic pulses can facilitate the mapping of through-bond connectivities. The method employs a suitable phasing scheme for generating the desired isotropic mixing Hamiltonian and requires moderate (13)C RF field strength only.
Carbon Isotopes↗
Adiabatic heteronuclear decoupling in rotating solids.
In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.
Histidine↗
Broadband RFDR with adiabatic inversion pulses.
With a view to obtain (13)C chemical shift correlation spectra of uniformly labelled peptides/proteins at high magnetic fields and high magic angle spinning frequencies (omega(r)/2pi </= 20 kHz), the efficacy of RFDR with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements employing different adiabatic pulse phasing schemes, shapes and durations. It is demonstrated that homonuclear dipolar recoupling with superior performance under resonance offset and H(1) inhomogeneity effects and without strong dependence on the (13)C chemical shift differences can be achieved with adiabatic pulses. It is shown that (13)C chemical shift correlation spectra in the entire range of carbon chemical shifts can be obtained efficiently with short adiabatic inversion pulses. In situations where correlation spectra of only the aliphatic region are required, the possibility for minimising the interference between the recoupling and decoupling RF fields with long adiabatic pulses, at low recoupling power levels and without compromising the broadband RFDR characteristics, is also indicated.
Amino Acid Sequence↗
REDOR: an assessment of the efficacy of dipolar recoupling with adiabatic inversion pulses.
A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3-6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model 15N-13C spin systems, (13C',15N) Aib-(15N) Aib-NH2 (Aib = alpha-aminoisobutyric acid) and (1-13C,15N) glycine.
Carbon Isotopes↗
Chemical shift correlation via RFDR: elimination of resonance offset effects.
It is shown that it is possible to effectively execute RFDR experiments with adiabatic inversion pulses and obtain resonance offset compensation that is superior to what can be achieved by conventional rectangular pulses. Employing 40-micros tanh/tan adiabatic pulses at a power level of approximately 38 kHz and a spinning speed of 12 kHz it is demonstrated that the range of resonance offset compensation achieved is sufficient to generate, via a single experiment, homonuclear chemical shift correlation spectra in the entire (13)C chemical shift range in peptides/proteins at the currently available field strengths.
Amino Acids↗