Isotope labeling for studying RNA by solid-state NMR spectroscopy

Abstract

Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.

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

“…In the past few years, we have proposed a route to achieve the assignment of RNA resonances as well as to obtain structural restraints for RNA‐structure determination by ssNMR . 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 .…”

“…From the 2D 13 C, 13 C SPC5 3 spectra of the RNP complexes assembled with non‐isotope labeled SL‐L7Ae and nucleotide‐type specific labeled RNA (Figure E,F and Supporting Information, Figure S7), we obtained 72 V para / V dia values (24 for each of the SL‐L7Ae‐K32C–RNA, SL‐L7Ae‐N38C–RNA, and SL‐L7Ae‐I93C–RNA complexes). These values were converted into distance ranges using the PRE‐to‐distance converter established from the intramolecular PRE data and the procedure shown in Figure S6 in the Supporting Information.…”

Characterizing Hydrogen Bonds in Intact RNA from MS2 Bacteriophage Using Solid State Magic Angle Spinning NMR

“…Large RNA molecules (>100nt) are challenging for solution NMR due to high spectral overlap and for larger sizes, due to decreased relaxation times. Such limitations are partially solved by segmentally labelling the RNA to reduce the spectral congestion (12, 13). They are also hard to obtain by crystallography techniques, and thus advanced sequence-based algorithms are used to predict their secondary structure.…”

Identification of weak non-canonical base pairs around riboswitch-ligand recognition sites by solid-state NMR exchange spectroscopy

“…In recent years, solid-state NMR (SSNMR) has emerged rapidly as an important tool in RNA studies. [19][20][21][22][23][24][25][26][27][28][29] This approach has unique advantages for studying RNAs across a broad range of molecular sizes, 23,24,27 and has shown great potential in studies of RNA aggregates, i.e., those involved in liquid-liquid phase separation systems. 30 In particular, the SSNMR-based hydrogen-deuterium exchange has been used to characterize RNA-protein interactions.…”