Enriching s4U‐RNA Using Methane Thiosulfonate (MTS) Chemistry

Erin E. Duffy1, Matthew D. Simon2

1 Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, 2 Chemical Biology Institute, Yale University, West Haven, Connecticut
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.12
Online Posting Date:  December, 2016
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Abstract

Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4‐Thiouridine (s4U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s4U‐RNA is the efficiency of the chemistry used to enrich s4U‐RNA. Here, we present a protocol for s4U‐RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s4U‐RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS‐biotin), followed by enrichment on streptavidin beads. The efficiency of this chemistry reduces enrichment bias and requires less starting material, thereby expanding the utility of s4U to study RNA biology. © 2016 by John Wiley & Sons, Inc.

Keywords: metabolic labeling; 4‐thiouridine; methane thiosulfonate; RNA metabolism

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cultured cells (e.g., HEK293T cells; at least 1 × 106)
  • RPE1 medium (see recipe)
  • 4‐thiouridine (s4U; MP Biomedicals, cat. no. 0215213405
  • TRIzol reagent (Life Technologies, cat. no 15596‐026)
  • Chloroform (AmericanBio, cat. no. AB00350‐00500)
  • 100% isopropanol
  • Dithiothreitol (DTT; VWR International, cat. no. EM‐3860)
  • 20 mg/ml glycogen (RNase‐free; AmericanBio, cat. no AB00670‐00020)
  • 100% and 75% ethanol, RNA‐grade
  • DEPC‐treated water (Life Technologies, cat. no. AM9906)
  • TURBO DNase (Life Technologies, cat. no. AM2238) and10× TURBO DNase reaction buffer
  • 3 M sodium acetate, pH 5.5
  • 25:24:1 (v/v/v) phenol:chloroform:isoamyl alcohol, pH 7.7 to 8.3 (Sigma Aldrich, cat. no. 77617‐100 ML)
  • 2× RNA fragmentation buffer (see recipe)
  • 250 mM and 0.5 M disodium EDTA (Sigma Aldrich, cat. no. E5134‐500 G)
  • RNeasy Mini Kit (Qiagen, cat. no. Q74106)
  • 2‐mercaptoethanol (Sigma Aldrich, cat. no. M3148‐25 ML)
  • MTSEA‐biotin‐XX (Biotium, cat. no. 89139‐636)
  • Dimethyl formamide (J.T. Baker, cat. no. 9344‐13)
  • 1 M HEPES, pH 7.4
  • 24:1 chloroform:isoamyl alcohol (Fisher Scientific, cat. no. 3160‐450 ML)
  • High‐salt wash buffer
  • Bead‐blocking buffer (see recipe)
  • Dynabeads MyOne Streptavidin C1 magnetic beads (Life Technologies, cat. no. 65001)
  • Elution buffer (see recipe)
  • Qubit RNA HS assay kit (Thermo Fisher Scientific, cat. no. Q32855)
  • Ambion century plus RNA marker (Thermo Fisher Scientific, cat. no. AM7145)
  • MAXIscript T7 transcription kit (Thermo Fisher Scientific, cat. no. AM1312)
  • Cy5‐CTP (GE Healthcare, cat. no. 25‐8010‐87)
  • 4‐thiouridine‐5′‐triphosphate (s4UTP; Trilink Bio, cat. no. N‐1025)
  • SequaGel UreaGel 29:1 Denaturing Gel System (National Diagnostics, cat. no. EC‐829)
  • Ammonium persulfate
  • TEMED
  • 1× TBE buffer (see recipe)
  • 6‐well culture plate
  • Refrigerated centrifuge
  • Gel‐loading tips
  • Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific, cat. no. ND‐2000c)
  • Phase‐lock gel tubes, heavy, 1.5‐ml (5‐Prime, cat. no. FP2302820)
  • UV‐vis spectrophotometer
  • 94°C heat block
  • Rotator (Glas‐Col, cat. no. 099 A MR1512)
  • MagRack 6 magnetic stand (GE Healthcare, cat. no. 28948964)
  • PCR tube strips
  • DynaMag 96 side magnet (magnetic rack for 96‐well plates; Life Technologies, cat. no. 12331D)
  • Qubit 3.0 Fluorometer (Thermo Fisher Scientific, cat. no. Q33216)
  • 15‐ml conical tubes (e.g., Corning Falcon)
  • Gel cassettes 1.0 mm (Life Sciences, cat. no. NC2010)
  • 10‐well comb
  • Typhoon FLA 9500 gel scanner (GE Healthcare) with Cy5 fluorescence filters
  • Additional reagents and equipment for gel electrophoresis (Gallagher, )
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Figures

Videos

Literature Cited

Literature Cited
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