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).

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Expansions of short nucleotide sequence repeats are associated with a number of neuromuscular diseases. [1][2][3] CTG triplet repeat expansions in the 3' noncoding region of the myotonic dystrophy protein kinase (DMPK) gene give rise to transcripts harboring CUG triplet repeat expansions on the RNA level. Repeats containing > 50 CUG triplets cause in trans-dominant fashion the most frequent form of adult-onset muscular dystrophy (DM1). The current model for DM1 pathogenesis strongly suggests that such repeats fold into large stable double-stranded RNA hairpins, which bind and sequester muscleblind (MBNL) proteins that are involved in the alternative splicing of a number of pre-mRNAs. As a consequence, the MBNL proteins are unavailable to the splicing machinery, and a number of important muscular premRNAs, for example, for the chloride channel ClC-1 and the insulin receptor, are aberrantly spliced; this process ultimately leads to the clinical manifestations of DM1.To decipher the structural basis of DM1, which could potentially permit the development of suitable drugs that would interfere with the sequestration of MBNL proteins by CUG repeats, we have recently initiated, as a first step, a magic-angle-spinning (MAS) solid-state NMR study of a % 100-kDa RNA composed of 97 CUG repeats ((CUG) 97 ). 15 N-15 N chemical-shift correlation experiments have enabled us to show the presence of canonical GC base pairs in this RNA. [4] In addition, analysis of the observed 13 C chemical shifts for the sugar carbon atoms suggested that (CUG) 97 adopts an A-form helix conformation with a C3'-endo sugar pucker and an anti conformation of the glycosidic torsion angle c.[5] Herein, we have explored the possibility of obtaining structural information directly by exploiting the dependence of inter-and intranucleotide 1 H-1 H distances on RNA conformation. Since the extraction of 1 H-1 H distances by direct 1 H observation techniques is still difficult in the solid state, we have utilized in this study the potential of protonproton dipolar-coupling-mediated chemical-shift correlation spectroscopy of low-gamma nuclei [6][7][8][9][10][11][12][13] for mapping the spatial proximity of the sugar and aromatic protons in (CUG) 97 .The radio frequency (RF) pulse sequence [13] and a schematic representation of the double-stranded (CUG) 97 employed are shown in Figure 1. Longitudinal 1 H magnetization exchange mediated by proton-proton dipolar coupling is allowed to take place during the spin diffusion period t mix . The experiment is carried out with a very short CP contact time ( % 100 ms) and a proton spin diffusion mixing time, t mix , of 100-200 ms. This time regime minimizes relayed magnetization transfers during the CP and t mix periods and, hence, crosspeaks with appreciable intensities are expected only between proton-attached 13 C sites that are connected by 1 H-1 H distances of less than % 3 . [8][9][10][11][12][13] 13 C homonuclear isotropic chemical-shift correlation spectra of a uniformly { 15 N, 13 C}-labeled sample of (CUG)...

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.

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.