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 13C-detected experiments for ribose assignment and amino groups, the CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the 15N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiment. © 2020 The Authors. Basic Protocol 1: Preparation of isotope-labeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RP-HPLC purification. Alternate Protocol 1: Purification of isotope-labeled RNA from in vitro transcription with preparative PAGE. Alternate Protocol 2: Purification of isotope-labeled RNA samples from in vitro transcription via centrifugal concentration. Support Protocol 1: Preparation of DNA template from plasmid. Support Protocol 2: Preparation of PCR DNA as template. Support Protocol 3: Preparation of T7 RNA Polymerase (T7 RNAP). Support Protocol 4: Preparation of yeast inorganic pyrophosphatase (YIPP). Basic Protocol 2: Preparation of site-specific labeled RNAs using a chemo-enzymatic synthesis. Support Protocol 5: Synthesis of modified nucleoside 3′,5′-bisphosphates. Support Protocol 6: Preparation of T4 RNA Ligase 2. Support Protocol 7: Setup of NMR spectrometer for heteronuclear-detected NMR experiments. Support Protocol 8: IPAP and DIPAP for homonuclear decoupling. Basic Protocol 3: 13C-detected 3D (H)CC-TOCSY, (H)CPC, and (H)CPC-CCH-TOCSY experiments for ribose assignment. Basic Protocol 4: 13C-detected 2D CN-spin filter HSQC experiment. Basic Protocol 5: 13C-detected C(N)H-HDQC experiment for the detection of amino groups. Support Protocol 9: 13C-detected CN-HSQC experiment for amino groups. Basic Protocol 6: 13C-detected “amino”-NOESY experiment. Basic Protocol 7: 15N-detected BEST-TROSY experiment.