大功能rna的核磁共振波谱:从样品制备到低伽马检测

Q4 Chemistry

Current Protocols in Nucleic Acid Chemistry Pub Date : 2020-09-22 DOI:10.1002/cpnc.116

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

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引用次数: 4

摘要

核磁共振波谱是rna结构和生物物理表征的有效方法。核磁共振波谱的应用受到RNA大小的限制，通常需要同位素标记甚至选择性标记的RNA。此外，还介绍了新的核磁共振脉冲序列，如异核探测核磁共振实验。本文提供了通过体外转录制备同位素标记RNA用于核磁共振光谱的详细方案。该方案涵盖了所有步骤，从DNA模板的制备到毫克RNA数量的转录。此外，我们提出了一种化学酶方法，在任何RNA的任何位置引入单个修饰的核苷酸。关于核磁共振方法，我们分享了一套异核检测核磁共振实验的实施方案，包括13c检测核糖分配和氨基的实验，cn -自旋滤波器异核单量子相干(HSQC)检测亚胺基团和15n检测带选择性激发短瞬态横向弛豫优化光谱(BEST-TROSY)实验。©2020作者。基本方案1:使用T7 RNAP, DEAE色谱法和RP-HPLC纯化体外转录的同位素标记RNA样品备用方案1:使用制备试剂纯化体外转录的同位素标记RNA样品备用方案2:通过离心浓缩纯化体外转录的同位素标记RNA样品支持方案1:从质粒制备DNA模板支持方案2:制备PCR DNA作为模板支持方案3:制备T7 RNA聚合酶(T7 RNAP)支持方案4:制备酵母无机焦磷酸酶(YIPP)基本方案2:使用化学酶合成制备位点特异性标记RNA支持方案5:合成修饰核苷3 '，5 ' -二磷酸盐支持方案6:制备T4 RNA连接酶2支持方案7:设置核磁共振波谱仪用于异核检测核磁共振实验支持方案8:IPAP和DIPAP用于同核解耦基本协议3:13c检测3D (H)CC-TOCSY， (H)CPC和(H)CPC- cch - tocsy实验用于核糖分配基本协议4:13c检测2D cn -自旋过滤器HSQC实验基本协议5:13c检测C(N)H- hdqc实验用于检测氨基支持协议9:13c检测CN-HSQC实验用于检测氨基基本协议6:13c检测“氨基”noesy实验基本协议7:15n检测BEST-TROSY实验

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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

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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 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. © 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: 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

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Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry

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期刊介绍： Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.