Robbin Schnieders, Bozana Knezic, Heidi Zetzsche, Alexey Sudakov, Tobias Matzel, Christian Richter, Martin Hengesbach, Harald Schwalbe, Boris Fürtig
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Regarding NMR methodology, we share protocols for the implementation of a suite of heteronuclear-detected NMR experiments including <sup>13</sup>C-detected experiments for ribose assignment and amino groups, the CN-spin filter heteronuclear single quantum coherence (HSQC) for imino groups and the <sup>15</sup>N-detected band-selective excitation short transient transverse-relaxation-optimized spectroscopy (BEST-TROSY) experiment. © 2020 The Authors.</p><p><b>Basic Protocol 1</b>: Preparation of isotope-labeled RNA samples with in vitro transcription using T7 RNAP, DEAE chromatography, and RP-HPLC purification</p><p><b>Alternate Protocol 1</b>: Purification of isotope-labeled RNA from in vitro transcription with preparative PAGE</p><p><b>Alternate Protocol 2</b>: Purification of isotope-labeled RNA samples from in vitro transcription via centrifugal concentration</p><p><b>Support Protocol 1</b>: Preparation of DNA template from plasmid</p><p><b>Support Protocol 2</b>: Preparation of PCR DNA as template</p><p><b>Support Protocol 3</b>: Preparation of T7 RNA Polymerase (T7 RNAP)</p><p><b>Support Protocol 4</b>: Preparation of yeast inorganic pyrophosphatase (YIPP)</p><p><b>Basic Protocol 2</b>: Preparation of site-specific labeled RNAs using a chemo-enzymatic synthesis</p><p><b>Support Protocol 5</b>: Synthesis of modified nucleoside 3′,5′-bisphosphates</p><p><b>Support Protocol 6</b>: Preparation of T4 RNA Ligase 2</p><p><b>Support Protocol 7</b>: Setup of NMR spectrometer for heteronuclear-detected NMR experiments</p><p><b>Support Protocol 8</b>: IPAP and DIPAP for homonuclear decoupling</p><p><b>Basic Protocol 3</b>: <sup>13</sup>C-detected 3D (H)CC-TOCSY, (H)CPC, and (H)CPC-CCH-TOCSY experiments for ribose assignment</p><p><b>Basic Protocol 4</b>: <sup>13</sup>C-detected 2D CN-spin filter HSQC experiment</p><p><b>Basic Protocol 5</b>: <sup>13</sup>C-detected C(N)H-HDQC experiment for the detection of amino groups</p><p><b>Support Protocol 9</b>: <sup>13</sup>C-detected CN-HSQC experiment for amino groups</p><p><b>Basic Protocol 6</b>: <sup>13</sup>C-detected “amino”-NOESY experiment</p><p><b>Basic Protocol 7</b>: <sup>15</sup>N-detected BEST-TROSY experiment</p>","PeriodicalId":10966,"journal":{"name":"Current Protocols in Nucleic Acid Chemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpnc.116","citationCount":"4","resultStr":"{\"title\":\"NMR Spectroscopy of Large Functional RNAs: From Sample Preparation to Low-Gamma Detection\",\"authors\":\"Robbin Schnieders, Bozana Knezic, Heidi Zetzsche, Alexey Sudakov, Tobias Matzel, Christian Richter, Martin Hengesbach, Harald Schwalbe, Boris Fürtig\",\"doi\":\"10.1002/cpnc.116\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>NMR spectroscopy is a potent method for the structural and biophysical characterization of RNAs. 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引用次数: 4
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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 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. © 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
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