RNA Structure Analysis of Viruses Using SHAPE

Cecily P. Burrill1, Raul Andino1

1 University of California, San Francisco, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15H.3
DOI:  10.1002/9780471729259.mc15h03s30
Online Posting Date:  October, 2013
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Abstract

Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) provides a means to investigate RNA structure with better resolution and higher throughput than has been possible with traditional methods. We present several protocols, which are based on a variety of previously published methods and were adapted and optimized for the analysis of poliovirus RNA in the Andino laboratory. These include methods for nondenaturing RNA extraction, RNA modification and primer extension, and data processing in ShapeFinder. Curr. Protoc. Microbiol. 30:15H.3.1‐15H.3.12. © 2013 by John Wiley & Sons, Inc.

Keywords: RNA structure; selective 2′ hydroxyl acylation; primer extension; SHAPE

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Nondenaturing RNA Extraction
  • Basic Protocol 2: Selective 2′ Hydroxy Acylation Analyzed by Primer Extension (SHAPE)
  • Alternate Protocol 1: Folding and Modification of In Vitro Transcribed RNA
  • Basic Protocol 3: Processing and Analysis of SHAPE Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Nondenaturing RNA Extraction

  Materials
  • 20% (w/v) sodium dodecyl sulfate (SDS; appendix 2A)
  • 10 mg/ml proteinase K
  • RNA source material (e.g., purified virions or cell lysate)
  • Tris‐saturated phenol, pH 7.4 (see recipe for buffered phenol in appendix 2A)
  • 3 M NaCl
  • 100% ethanol
  • RNA modification buffer (see recipe)
  • NanoDrop spectrophotometer (http://www.nanodrop.com)

Basic Protocol 2: Selective 2′ Hydroxy Acylation Analyzed by Primer Extension (SHAPE)

  Materials
  • RNA of interest in RNA modification buffer ( protocol 1)
  • Dimethyl sulfoxide (DMSO)
  • 32.5 mM N‐methylisotoic anhydride (NMIA; Invitrogen, cat. no. M‐25) in DMSO
  • 50 mM EDTA
  • 20 µg/ml glycogen
  • 3 M NaCl
  • 100% ethanol
  • 0.5× TE buffer, pH 8.0 (see recipe)
  • In vitro–transcribed RNA of interest in 0.5× TE buffer, pH 8.0 (for sequencing/alignment reactions)
  • 1 µM oligo 5′‐tagged with fluorophore 1
  • 1 µM oligo 5′‐tagged with fluorophore 2
  • 5× First‐strand buffer (supplied with SuperScript III)
  • 0.1 M DTT (supplied with SuperScript III)
  • dNTP mix (10 mM each dNTP; appendix 2A)
  • 10 mM ddATP
  • 10 mM ddCTP
  • Nuclease‐free water
  • SuperScript III (Invitrogen)
  • 1 M NaOH
  • 1 M HCl
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 75% (v/v) ethanol
  • HiDi formamide (Applied Biosystems)
  • Thermal cycler
  • Additional reagents and equipment for capillary electrophoresis (Smith and Nelson, )

Alternate Protocol 1: Folding and Modification of In Vitro Transcribed RNA

  Materials
  • In vitrotranscribed RNA of interest in 0.5× TE buffer, pH 8.0
  • 0.5× TE buffer, pH 8.0 ( appendix 2A)
  • 3.3× RNA folding buffer (see recipe)
  • Dimethyl sulfoxide (DMSO)
  • 32.5 mM N‐methylisotoic anhydride (NMIA; Invitrogen, cat. no. M‐25) in DMSO
  • 50 mM EDTA
  • 20 µg/ml glycogen
  • 3 M NaCl
  • 100% ethanol
  • 95°C water bath or heat block
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Figures

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Literature Cited

Literature Cited
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