Rapid access to RNA resonances by proton-detected solid-state NMR at >100 kHz MAS

Abstract

Fast (>100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Here we apply this technique to RNA and assign nucleotide spin systems through highly sensitive multidimensional experiments.

Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

“…In the case of protein–RNA PREs, there are several technical problems linked to this approach. First, in our previous work we measured bulk coherence lifetimes (T 2 ′) of pyrimidine C6 and purine C8 at different MAS rates and obtained values of 5–6 ms and 11 ms at MAS rates of 40 and 100 kHz, respectively. These short values suggest that the measurement of transversal relaxation rates for these sites with acceptable signal‐to‐noise ratio would require long measurement times .…”

“…The strategy relies on nucleotide‐type selective 13 C, 15 N‐labeling . In more recent work, we have extended ssNMR proton detection at fast magic angle spinning (MAS) rates to RNA and demonstrated the assignment of ribose resonances in a uniformly labeled sample . These experiments, together with those developed by several other laboratories to yield the structure of proteins, permit the structure determination of the RNA and protein parts of an RNP complex by ssNMR.…”

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

“…Finally,o ur work showedt hat the LHSQC scheme was applicable to fractional deuterated RNAst od etect the rigid segments.I ti sw orth noting that 1 H-detected SSNMR spectroscopy on RNA is an intriguing direction towards RNA characterization. [28,31] To the best of our knowledge, this is the first proven example of J-based 1 H-detected HSQC of RNA by fast MAS. Because HSQC experimentsa re the basic setup for resonance assignments, it is anticipated that fast 3D SSNMR experiments could be designed by involving LHSQCw ith the nonuniform sampling scheme [62][63][64][65][66] or projected NMR scheme [67,68] to facilitate RNA assignments under fast MAS conditions.…”

“…Finally, our work showed that the LHSQC scheme was applicable to fractional deuterated RNAs to detect the rigid segments. It is worth noting that 1 H‐detected SSNMR spectroscopy on RNA is an intriguing direction towards RNA characterization . To the best of our knowledge, this is the first proven example of J‐ based 1 H‐detected HSQC of RNA by fast MAS.…”

“…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.…”