Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy

Abstract

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).

The structural basis of myotonic dystrophy from the crystal structure of CUG repeats

“…1C) similar to expanded CUG repeats found in the dmpk gene in patients with DM1. The formation of WatsonCrick G-C base pairs was also observed in NMR studies of r(CUG) 97 (10,34). This agreement suggests that the crystal structure of r(CUG) 6 is likely relevant to the stem of the stem loops that long CUG repeats form.…”

Constraints on the Structure of (CUG)97 RNA from Magic‐Angle‐Spinning Solid‐State NMR Spectroscopy

“…[4] A hydrated, undiluted { 15 N, 13 C}-labeled RNA sample of (CUG) 97 [4] (8.5 mg) and a hydrated, specifically [ …”

“…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) 97 obtained with different short proton spin diffusion mixing times are shown in Figure 2. The resonance assignments indicated are based on our recent studies.…”

“…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 .…”