Open Access

Robbin Schnieders, Božana Knežić, Heidi Zetzsche, Alexey Sudakov, Tobias Matzel, Christian Richter, Martin Hengesbach, Harald Schwalbe, Boris Fürtig

• 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. 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