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

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 C-detected experiments for ribose assignment and amino groups, the CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the N-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: C-detected 3D (H)CC-TOCSY, (H)CPC, and (H)CPC-CCH-TOCSY experiments for ribose assignment Basic Protocol 4: C-detected 2D CN-spin filter HSQC experiment Basic Protocol 5: C-detected C(N)H-HDQC experiment for the detection of amino groups Support Protocol 9: C-detected CN-HSQC experiment for amino groups Basic Protocol 6: C-detected "amino"-NOESY experiment Basic Protocol 7: N-detected BEST-TROSY experiment.