NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low‐Gamma Detection

Abstract

NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. The application of NMR spectroscopy is restricted in RNA size and most often requires isotope-labeled or even selectively labeled RNAs. Additionally, new NMR pulse sequences, such as the heteronuclear-detected NMR experiments, are introduced. We herein provide detailed protocols for the preparation of isotope-labeled RNA for NMR spectroscopy via in vitro transcription. This protocol covers all steps, from the preparation of DNA template to the transcription of milligram RNA quantities. Moreover, we present a protocol for a chemo-enzymatic approach to introduce a single modified nucleotide at any position of any RNA. Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear-detected NMR experiments including 13 C-detected experiments for ribose assignment and amino groups, the CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15 N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiment.

1H, 13C and 15N chemical shift assignment of the stem-loops 5b + c from the 5′-UTR of SARS-CoV-2

“…The recombinant vectors pHDV-5_SL5b + c and pHDV-5_ SL5b_GC were transformed and amplified in the Escherichia coli strain DH5α. Plasmid-DNA was purified with a large scale DNA isolation kit (Gigaprep; Qiagen) according to the manufacturer's instructions and linearized with Hin-dIII prior to in-vitro transcription by T7 RNA polymerase [P266L mutant, prepared as described in (Guillerez et al 2005;Schnieders et al 2020)]. 15 ml transcription reactions [20 mM DTT, 2 mM spermidine, 200 ng/µl template, 200 mM Tris/glutamate (pH 8.1), 40 mM Mg(OAc) 2 , 12 mM NTPs, 32 µg/ml T7 RNA Polymerase, 20% DMSO (b + c) or 0% DMSO (b_GC)] were performed to obtain sufficient amount of RNA.…”

Incorporation and Utility of a Responsive Ribonucleoside Analogue in Probing the Conformation of a Viral RNA Motif by Fluorescence and 19F NMR Spectroscopy

“…[1][2][3][4][5] Basic understanding of RNA folding and its interaction with metal ions, proteins, nucleic acids and metabolites has not only provided valuable information on structure-function relationship but has also provided means to target RNA sequences related to disease states. [6][7][8][9] Biophysical techniques like fluorescence, [10][11][12][13] NMR spectroscopy, [14][15][16] EPR, [17,18] and X-ray crystallography [19,20] are routinely used to study RNA structure, dynamics and functions in real time and atomic level. Notably, fluorescencebased methods employing RNA ONs labelled with fluorescent nucleoside probes significantly aid the study of RNA.…”

Time-resolved NMR-spectroscopic studies of conformational dynamics in DNA G-quadruplexes

“…[576][577][578][579][580][581] A manifold of homo-and heteronuclear NMRexperiments has been developed that help for the assignment of the relevant proton (in particular N-H1 imino proton) signals. [582][583][584] In addition to that, many NMR-experiments are available that yield information on base pair patterns or structural restraints on dihedral angles (via scalar couplings, abbr.…”

“…The strategies and advantages for these experimental approaches have been extensively reviewed. [582][583][584] In particular for DNA G-quadruplexes, NMR is the most important method for structure determination at atomic resolution. 310,585,586 From 277 PDB-structures ("G-quadruplex and DNA", December 2020) 154 (56%) have been solved by NMR and 123 (44%) by X-ray crystallography (NMR for: "nucleic acids": 38%; total: 8%).…”

“…However, most heteronuclear 2D-correlated experiment relies on isotope enrichment for the NMR-active (spin-½) nuclei 13 C and 15 N. Isotope labelled RNA (both selectively and uniformly) is easily accessible via in vitro transcription using T7 RNA-Polymerase and the respective isotope labeled NTPs. 582,584 Preparation of isotope labeled, single stranded DNA via enzymatic methods in appropriate amounts for NMR investigations is not routinely established. For DNA oligonucleotides, isotope labeling is introduced typically via solid-phase oligonucleotide synthesis.…”