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

Abstract

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)...

Structural studies of CNG repeats

“…The earliest paper presents an analysis of a 97-long run of CUG repeats in solid state (75). The authors noted the presence of canonical C-G pairs and observed resonances consistent with an A-form helix with a C3′- endo sugar pucker and an anti conformation of the glycosidic torsion angle.…”

Structure and Dynamics of RNA Repeat Expansions That Cause Huntington’s Disease and Myotonic Dystrophy Type 1

“…A solid state NMR study of a construct with 97 r(CUG) repeats revealed A-form geometry with C3′- endo geometry and an anti glycosidic torsion angle (χ). (56) NMR spectroscopy can also provide information about dynamics, which may be used to design therapeutics to target these RNA repeats. (57) Although conformations of RNAs in solution would not be influenced by crystal packing, signal overlap may complicate full assignment of resonances.…”

Longitudinal Relaxation Optimization Enhances 1H‐Detected HSQC in Solid‐State NMR Spectroscopy on Challenging Biological Systems

“…Solid-state (SS) NMR spectroscopy has been used extensively to study challenging biological systems, [1][2][3][4][5] including large transmembrane proteins; [6][7][8][9][10][11][12][13][14] membrane fusion polypeptides; [15][16][17] protein fibrils; [18][19][20][21][22] native collagens; [23,24] polysaccharides; [25][26][27] and, more recently,R NA. [28][29][30][31][32][33][34] The application of SSNMR spectroscopy to in vivo studies is also an attractive field, [35][36][37] whichp rovides great opportunities for characterizing the cellular components of living cells [37,38] and investigating the effects of antibiotics on cells. [39,40] Nevertheless, owing to the intrinsic weakness of low sensitivity,i to ften takes al ong time to obtain as atisfactory signal-to-noise (S/N)r atio of the SSNMR spectra;t hus making it difficult to investigate biological samples with as hort lifetime or those difficult to obtain in large quantities.…”