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.
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.
Authors: Klara R Mertinkus; J Tassilo Grün; Nadide Altincekic; Jasleen Kaur Bains; Betül Ceylan; Jan-Peter Ferner; Lucio Frydman; Boris Fürtig; Martin Hengesbach; Katharina F Hohmann; Daniel Hymon; Jihyun Kim; Božana Knezic; Mihajlo Novakovic; Andreas Oxenfarth; Stephen A Peter; Nusrat S Qureshi; Christian Richter; Tali Scherf; Andreas Schlundt; Robbin Schnieders; Harald Schwalbe; Elke Stirnal; Alexey Sudakov; Jennifer Vögele; Anna Wacker; Julia E Weigand; Julia Wirmer-Bartoschek; Maria A Wirtz Martin; Jens Wöhnert Journal: Biomol NMR Assign Date: 2022-02-18 Impact factor: 0.731