Probing RNA Structure with Chemical Reagents and Enzymes

William A. Ziehler1, David R. Engelke1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.1
DOI:  10.1002/0471142700.nc0601s00
Online Posting Date:  May, 2001
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Abstract

This unit provides thorough coverage of the most useful chemical and enzyme probes that can be used to examine RNA secondary and tertiary structure. Footprinting methods are presented using dimethyl sulfate, diethyl pyrocarbonate, ethylnitrosourea, kethoxal, CMCT, and nucleases. For chemical probes, both strand scission and primer extension detection protocols are included.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Modification and Cleavage of RNA Using Dimethyl Sulfate
  • Alternate Protocol 1: Modification and Cleavage of RNA Using Diethylpyrocarbonate
  • Alternate Protocol 2: Modification and Cleavage of RNA Using Ethylnitrosourea
  • Basic Protocol 2: Cleavage of RNA Using Nucleases
  • Support Protocol 1: Labeling the 5′ RNA Terminus Using T4 Polynucleotide Kinase and [γ‐32P]ATP
  • Support Protocol 2: Labeling the 3′ RNA Terminus Using T4 RNA Ligase and [32P]pCp
  • Support Protocol 3: Preparing Standards for Electrophoresis of End‐Labeled RNA Cleavage Products
  • Basic Protocol 3: Modification of RNA at Watson‐Crick Positions Using Dimethyl Sulfate
  • Alternate Protocol 3: Modification of RNA at Watson‐Crick Positions Using Kethoxal
  • Alternate Protocol 4: Modification of RNA at Watson‐Crick Positions Using CMCT
  • Support Protocol 4: Detection of RNA Cleavage or Modification by Primer Extension
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Modification and Cleavage of RNA Using Dimethyl Sulfate

  Materials
  • 1 M HEPES, pH 7.8
  • 1 M KCl
  • 0.1 M MgCl 2
  • 1 µg/µL carrier RNA (see recipe)
  • Sample RNA, end labeled (see protocol 5 or protocol 6) or unlabeled
  • RNase‐free water (see recipe)
  • ≥99% dimethyl sulfate (DMS) (Aldrich)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% and 70% (v/v) ethanol
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 0.2 M NaBH 4, prepared fresh
  • 1 M aniline acetate buffer (see recipe)
  • FEXS solution (see recipe; for end‐labeled RNA)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (e.g., appendix 3B or CPMB UNIT ) or primer extension (see protocol 11)
NOTE: DMS is highly toxic and a suspected carcinogen; use appropriate precautions for handling, storage, and disposal.

Alternate Protocol 1: Modification and Cleavage of RNA Using Diethylpyrocarbonate

  • ≥97% diethylpyrocarbonate (DEPC) (store up to 1 year at 2° to 8°C)
NOTE: DEPC is toxic. Handle with appropriate care.

Alternate Protocol 2: Modification and Cleavage of RNA Using Ethylnitrosourea

  • ENU/ethanol solution (see recipe)
  • 0.1 M Tris⋅Cl, pH 9.0 ( appendix 2A)

Basic Protocol 2: Cleavage of RNA Using Nucleases

  Materials
  • One of the following nucleases (see Table 6.1.1):
    •  Mung bean nuclease
    •  RNase A
    •  RNase CL3
    •  RNase I(ONE)
    •  RNase Phy M
    •  RNase T1
    •  RNase T2
    •  RNase U2
    •  RNase V1
    •  S1 nuclease
  • 0.1 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • 1 M KCl ( appendix 2A)
  • 0.1 M MgCl 2
  • 1 µg/µL carrier RNA (see recipe)
  • Sample RNA, end labeled (see protocol 5 or protocol 6) or unlabeled
  • RNase‐free water (see recipe)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% and 70% (v/v) ethanol
  • FEXS solution (see recipe)
  • Additional reagents and equipment for direct gel electrophoresis (e.g., appendix 3B or CPMB UNIT ) or primer extension (see protocol 11)

Support Protocol 1: Labeling the 5′ RNA Terminus Using T4 Polynucleotide Kinase and [γ‐32P]ATP

  Materials
  • RNA of interest
  • 150 U/µL bacterial alkaline phosphatase (BAP; Life Technologies)
  • RNase‐free water (see recipe)
  • 10× dephosphorylation buffer: 100 mM Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% and 70% (v/v) ethanol
  • 10 mM EDTA ( appendix 2A)
  • 10× kinase buffer (see recipe)
  • 0.1 M dithiothreitol (DTT)
  • 150 µCi/µL [γ‐32P]ATP (6000 Ci/mmol; NEN Life Sciences)
  • 10,000 U/mL T4 polynucleotide kinase
  • Stop mix (see recipe)
  • Water baths, 37°C and 90° to 100°C
  • Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction ( appendix 2A)

Support Protocol 2: Labeling the 3′ RNA Terminus Using T4 RNA Ligase and [32P]pCp

  • 0.5 M HEPES, pH 7.9 at 50 mM
  • 0.1 M MgCl 2
  • 0.1 mg/mL bovine serum albumin (BSA)
  • 1 mM ATP
  • Dimethyl sulfoxide (DMSO)
  • 500 µM 5′‐[32P]cytidine‐3′,5′‐bisphosphate ([32P]pCp; unit 6.3 or Amersham)
  • 20 to 25 U/µL T4 RNA ligase

Support Protocol 3: Preparing Standards for Electrophoresis of End‐Labeled RNA Cleavage Products

  Materials
  • RNA of interest, 32P‐end‐labeled to 50,000 cpm (see protocol 5 or protocol 6)
  • 100% (v/v) ethanol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • Na 2CO 3/EDTA solution (see recipe)
  • CU buffer (see recipe)
  • RNase T1 (Life Technologies)
  • 2 µg/µL tRNA carrier (see recipe for carrier RNA)
  • CEU buffer (see recipe)
  • FEXS solution (see recipe)
  • 95°C water bath

Basic Protocol 3: Modification of RNA at Watson‐Crick Positions Using Dimethyl Sulfate

  Materials
  • 0.5 M HEPES, pH 7.8
  • 1 M KCl ( appendix 2A)
  • 0.1 M MgCl 2
  • 1 µg/µl RNA of interest
  • RNase‐free water (see recipe)
  • 10.56 M dimethyl sulfate (DMS; 99+%)
  • 100% and 70% (v/v) ethanol
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • Additional reagents and equipment for primer extension (see protocol 11)
CAUTION: DMS is highly toxic and a suspected carcinogen; appropriate precautions should be taken for handling, storage, and disposal.

Alternate Protocol 3: Modification of RNA at Watson‐Crick Positions Using Kethoxal

  • 4.27 M kethoxal stock (ICN or Research Organics)

Alternate Protocol 4: Modification of RNA at Watson‐Crick Positions Using CMCT

  • 0.5 M potassium borate, pH 8.0
  • 0.5 M 1‐cyclohexyl‐3‐(2‐morpholinoethyl)carbodiimide metho‐p‐toluenesulfonate (CMCT) in RNase‐free H 2O (stable for several weeks at –20°C; Aldrich)
  • 37°C water bath

Support Protocol 4: Detection of RNA Cleavage or Modification by Primer Extension

  Materials
  • [5′‐32P]DNA oligomer (see protocol 5) complementary to 3′ end of the sample RNA
  • Sample RNA, cleaved or modified (see Basic Protocols protocol 11 to protocol 83 and Alternate Protocols protocol 21 to protocol 104)
  • RNase‐free water (see recipe)
  • Sample RNA, uncleaved and unmodified
  • 25 mM 4dNTP mix (see dNTPs in appendix 2A)
  • 0.1 M dithiothreitol (DTT)
  • 5× first strand buffer (see recipe)
  • 200 U/µL Superscript II RNase H(−) Moloney murine leukemia virus reverse transcriptase (MMLV RT; Life Technologies; see for alternative RTs)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% and 70% (v/v) ethanol
  • FENXB solution (see recipe)
  • Dideoxynucleotide sequencing reactions (e.g., CPMB UNIT ) of cDNA for sample RNA
  • Sequencing gel: 6% (w/v) acrylamide (20:1 mono‐/bis‐), 8 M urea, 1× TBE buffer ( appendix 2A for TBE buffer)
  • Water bath or heating block, 42° and 70°C
  • Additional reagents and equipment for running a sequencing gel ( appendix 3B)
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Figures

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Literature Cited

Literature Cited
   England, T., Bruce, A., and Uhlenbeck, O. 1980. Specific labeling of 3′ termini of RNA with T4 RNA ligase. Methods Enzymol. 65:65‐74.
   Gautheret, D., Major, F., and Cedergren, R. 1990. Computer modeling and display of RNA secondary and tertiary structures. Methods Enzymol. 183:318‐330.
   Gorodkin, J., Heyer, L., and Stormo, G. 1997. Finding the most significant common sequence and structure motifs in a set of RNA sequences. Nucl. Acids Res. 25:3724‐3732.
   Knapp, G. 1989. Enzymatic approaches to probing of RNA secondary and tertiary structure. Methods Enzymol. 180:192‐212.
   Krol, A. Carbon, P. 1989. A guide for probing native small nuclear RNA and ribonucleoprotein structures. Methods Enzymol. 180:212‐227.
   Major, F., Turcotte, M., Gautheret, D., Lapalme, G., Fillion, E., and Cedergren, R. 1991. The combination of symbolic and numerical computation for three‐dimensional modeling of RNA. Science 253:1255‐1260.
   Malhotra, A., Gabb, H., and Harvey, S. 1993. Modeling large nucleic acids. Curr. Opin. Struct. Biol. 3:241‐246.
   Milligan, J.F. Uhlenbeck, O.C. 1989. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 180:51‐62.
   Peattie, D.A. Gilbert, W. 1980. Chemical probes for higher‐order structure in RNA. Proc. Natl. Acad. Sci. U.S.A. 77:4679‐4682.
   Walter, A.E., Turner, D.H., Kim, J., Lyttle, M.H., Muller, P., Matthews, D.H., and Zuker, M. 1994. Coaxial stacking of helixes enhances binding of oligoribonucleotides and improves predictions of RNA folding. Proc. Natl. Acad. Sci. U.S.A. 91:9218‐9222.
   Zuker, M. 1989. On finding all suboptimal foldings of an RNA molecule. Science 244:48‐52.
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