RNA Structure Analysis of Viruses Using SHAPE

互联网2013-12-31

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Abstract
Table of Contents
Materials
Literature Cited

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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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 vitro– transcribed 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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Literature Cited

Literature Cited
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