Probing RNA Folding Pathways by RNA Fingerprinting

Sarah A. Woodson1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.4
DOI:  10.1002/cpnc.36
Online Posting Date:  September, 2017
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Abstract

This unit provides protocols for using native polyacrylamide gel electrophoresis to distinguish folding and unfolding conformers of RNA. It is useful for studying conformers that can exchange in a period of minutes or seconds, and that are thus difficult to study by solution‐based methods. Conformers that have been separated and immobilized in the gel matrix can be used to study catalytic activity with or without being eluted from the gel. The method can be applied to a wide variety of catalytic RNAs and RNA‐protein complexes. © 2017 by John Wiley & Sons, Inc.

Keywords: RNA; fingerprinting; native polyacrylamide gel electrophoresis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Native Gel Electrophoresis of 32P‐Labeled or Dye‐Labeled RNA
  • Alternate Protocol 1: Native GEL Electrophoresis of Dye‐Labeled RNA
  • Basic Protocol 2: Assay of Catalytic Activity by Two‐Dimensional Gel Electrophoresis
  • Alternate Protocol 2: Elution of Spliced Products from Native Polyacrylamide Gels
  • Basic Protocol 3: Chemical Modification of RNA in a Native Polyacrylamide Gel
  • Support Protocol 1: In Vitro Synthesis of 32P‐Labeled Pre‐RNA with T7 RNA Polymerase
  • Support Protocol 2: Larger Scale In Vitro Synthesis of Unlabeled Pre‐RNA with T7 RNA Polymerase
  • Support Protocol 3: Detection of Modified Bases by Primer Extension
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Native Gel Electrophoresis of 32P‐Labeled or Dye‐Labeled RNA

  Materials
  • 70% ethanol
  • 40% (w/v) 29:1 acrylamide/bisacrylamide, 4°C
  • 10× THEM buffer (see recipe)
  • 10% (w/v) ammonium persulfate, 4°C
  • TEMED, 4°C
  • 5× folding buffer (see recipe8)
  • 32P‐labeled RNA or dye‐labeled RNA, desalted (see protocol 5)
  • 30 mM MgCl 2
  • 5× glycerol loading buffer (see recipe)
  • 20 × 20–cm glass plates with 0.5‐mm spacers and comb (1 or 2 sets)
  • Vacuum source and 50‐mL side‐arm flask (for degassing)
  • 20 × 20–cm vertical gel electrophoresis apparatus with recirculating cooling reservoir (e.g., ThermoFisher Owl Scientific Penguin 10DS or equivalent)
  • Refrigerated recirculating bath to connect to gel apparatus
  • Spatula or razor blade
  • 3MM filter paper (Whatman)
  • Vacuum gel drying apparatus
  • X‐ray film for autoradiography or phosphorescent imager
CAUTION: Acrylamide and N,N′‐methylenebisacrylamide are neurotoxins and suspected carcinogens. Preparation of solutions with these compounds should be performed in a well‐ventilated fume hood, and extreme precautions taken to minimize contact with solids or solutions.

Alternate Protocol 1: Native GEL Electrophoresis of Dye‐Labeled RNA

  Additional Materials (also see protocol 1)
  • In vitro transcribed RNA, with unstructured region suitable for binding oligonucleotides
  • Synthetic RNA oligonucleotide, labeled with Cy5, Cy3, Alexa 488, FAM or other appropriate dye (∼20 nt), free of unconjugated dye and desalted
  • HK buffer (see recipe)
  • Typhoon or other fluorescence image
  • Heat block or water bath
Hybridize RNA with dye‐labeled oligonucleotide and refold

Basic Protocol 2: Assay of Catalytic Activity by Two‐Dimensional Gel Electrophoresis

  Materials
  • [32P] ATP
  • 0.1 mM GTP in folding buffer (see recipe)
  • 2× urea loading buffer (see recipe)
  • 40% (w/v) 29:1 acrylamide/bisacrylamide, 4°C
  • 10× TBE buffer ( appendix 2A)
  • Urea
  • 10% (w/v) ammonium persulfate
  • TEMED
  • Bed of ice (size of gel)
  • Phosphor imager
  • Razor blades
  • Glass plates (e.g., 20 × 20 cm or larger)
  • Additional reagents and equipment for native gel electrophoresis (see protocol 1)

Alternate Protocol 2: Elution of Spliced Products from Native Polyacrylamide Gels

  • TEN buffer (see recipe)
  • 10 mg/mL carrier tRNA (Sigma)
  • 70% and 100% ethanol, –20°C
  • TE buffer, pH 7.5 ( appendix 2A)
  • Rocker or orbital mixer
  • 4% to 6% denaturing polyacrylamide gel (see protocol 102)

Basic Protocol 3: Chemical Modification of RNA in a Native Polyacrylamide Gel

  • Unlabeled RNA (see protocol 6; see also unit 6.1)
  • Dimethyl sulfate (DMS, Sigma‐Aldrich; obtain fresh and store at –20°C)
  • 50% and 100% ethanol
  • Stop buffer A (see recipe)
  • 70 mg/mL kethoxal (ICN Biochemicals or Research Organics) in 20% ethanol (store at –20°C)
  • Stop buffer B (see recipe)
  • N‐methylisatoic anhydride (NMIA) stock solution
  • Dimethyl sulfoxide (DMSO), anhydrous
  • Buffered phenol ( appendix 2A)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • Additional reagents and equipment for primer extension (see protocol 7) and for sequencing gels (unit 6.1 and appendix 3B)
CAUTION: Dimethyl sulfate (DMS) is a carcinogen and should be handled in a fume hood with gloves that are impermeable to nonpolar solvents.

Support Protocol 1: In Vitro Synthesis of 32P‐Labeled Pre‐RNA with T7 RNA Polymerase

  Materials
  • 1 µg/µL plasmid DNA containing a T7 promoter, digested with appropriate restriction enzyme(s) (e.g. Feinbaum, ; Tabor, ) or 0.2 μg/μL PCR product containing a T7 promoter
  • 10× T7 RNAP buffer (see recipe)
  • 20 mM spermidine⋅HCl (store at –20°C)
  • 10× NTPs “low A” (see recipe)
  • 10 to 20 µCi [α‐32P]ATP (store at –20°C)
  • 500 to 1000 U/µL T7 RNA polymerase (store at –20°C)
  • TE buffer, pH 7.5 ( appendix 2A) or another buffer
  • 30°C water bath
  • Gel‐filtration spin column (e.g., TE‐100, Clontech), 4°C
  • Liquid scintillation counter
  • Scintillation fluid and vials
NOTE: Buffers and reagents should be kept on ice after thawing. Steps 1 to 4 are carried out at 4°C except where noted.

Support Protocol 2: Larger Scale In Vitro Synthesis of Unlabeled Pre‐RNA with T7 RNA Polymerase

  Materials
  • 1 µg/µL digested plasmid DNA (10 µg total) or 5 µg total PCR product DNA
  • 10× T7 RNAP buffer (see recipe)
  • 1 M spermidine⋅HCl
  • 50 or 100 mM each of ATP, CTP, GTP, and UTP
  • ∼1000 U/µL T7 RNA polymerase
  • 0.5 M EDTA ( appendix 2A)
  • 3 M sodium acetate, pH 5.0 ( appendix 2A)
  • 100% ethanol
  • TEN buffer (see recipe)
  • 15‐mL sterile polypropylene culture tubes
  • Microconcentrators with appropriate MWCO (e.g., Amicon Ultra 2 mL centrifugal filters)

Support Protocol 3: Detection of Modified Bases by Primer Extension

  • 1 pmol/µL 5′ 32P‐labeled sequencing primer (unit 6.1)
  • Diluted RNA for primer extension (see protocol 4, step 19)
  • 250 nm unmodified RNA (see protocol 6)
  • 2.5 mM ddATP, ddCTP, ddGTP, and ddTTP solutions, in water
  • 5× first‐strand synthesis buffer (Invitrogen; see recipe)
  • 10 mM dNTP mix: 10 mM each of dATP, dCTP, dGTP, and dTTP in water
  • Superscript III reverse transcriptase (Invitrogen Life Technologies)
  • 2× formamide loading buffer (see recipe)
  • 55°, 65°, 85°, and 95°C water baths or heating blocks
  • Additional reagents and equipment for preparing and running 6% or 8% polyacrylamide sequencing gel ( appendix 3B)
NOTE: The following steps are carried out on ice except where indicated. Thawed reagents should kept on ice throughout.
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Figures

Videos

Literature Cited

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