PMID- 32960489
OWN - NLM
STAT- MEDLINE
DCOM- 20210621
LR  - 20210621
IS  - 1934-9289 (Electronic)
IS  - 1934-9270 (Linking)
VI  - 82
IP  - 1
DP  - 2020 Sep
TI  - NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low-Gamma 
      Detection.
PG  - e116
LID - 10.1002/cpnc.116 [doi]
AB  - 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. (c) 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: (13) C-detected 3D (H)CC-TOCSY, (H)CPC, 
      and (H)CPC-CCH-TOCSY experiments for ribose assignment Basic Protocol 4: (13) 
      C-detected 2D CN-spin filter HSQC experiment Basic Protocol 5: (13) C-detected 
      C(N)H-HDQC experiment for the detection of amino groups Support Protocol 9: (13) 
      C-detected CN-HSQC experiment for amino groups Basic Protocol 6: (13) C-detected 
      "amino"-NOESY experiment Basic Protocol 7: (15) N-detected BEST-TROSY experiment.
CI  - (c) 2020 The Authors.
FAU - Schnieders, Robbin
AU  - Schnieders R
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Knezic, Bozana
AU  - Knezic B
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Zetzsche, Heidi
AU  - Zetzsche H
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Sudakov, Alexey
AU  - Sudakov A
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Matzel, Tobias
AU  - Matzel T
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Richter, Christian
AU  - Richter C
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Hengesbach, Martin
AU  - Hengesbach M
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Schwalbe, Harald
AU  - Schwalbe H
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
FAU - Furtig, Boris
AU  - Furtig B
AD  - Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular 
      Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-University, Germany.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Curr Protoc Nucleic Acid Chem
JT  - Current protocols in nucleic acid chemistry
JID - 101287865
RN  - 63231-63-0 (RNA)
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Carbon-13 Magnetic Resonance Spectroscopy/methods
MH  - Chromatography, Liquid/methods
MH  - DNA/genetics
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Isotope Labeling
MH  - Nuclear Magnetic Resonance, Biomolecular/*methods
MH  - RNA/*chemistry
OTO - NOTNLM
OT  - NMR
OT  - RNA
OT  - heteronuclear detection
OT  - isotope labeling
OT  - large functional RNAs
EDAT- 2020/09/23 06:00
MHDA- 2021/06/22 06:00
CRDT- 2020/09/22 12:14
PHST- 2020/09/22 12:14 [entrez]
PHST- 2020/09/23 06:00 [pubmed]
PHST- 2021/06/22 06:00 [medline]
AID - 10.1002/cpnc.116 [doi]
PST - ppublish
SO  - Curr Protoc Nucleic Acid Chem. 2020 Sep;82(1):e116. doi: 10.1002/cpnc.116.