Probing RNA Structural Dynamics and Function by Fluorescence Resonance Energy Transfer (FRET)

Nils G. Walter1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.10
DOI:  10.1002/0471142700.nc1110s11
Online Posting Date:  February, 2003
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Abstract

Biological function of RNA is often mediated by cyclic switching between several (meta‐)stable arrangements of tertiary structure. Fluorophore labeling of RNA offers a unique view into these folding and conformational switching events, since a fluorescence signal is sensitive to its molecular environment and can be continuously monitored in real time to produce kinetic rate information. This unit focuses on the practical implications of using fluorescence resonance energy transfer (FRET) to probe RNA structural dynamics and function. FRET is a particularly powerful fluorescence technique since, in addition to kinetic data, it provides insights into the structural basis of a conformational rearrangement. Protocols describe how to postsynthetically label RNA for FRET and how to acquire and analyze FRET data. Support protocols describe methods for deprotecting synthetic RNA and for purifying RNA by gel electrophoresis and HPLC. Considerations for selecting appropriate RNA, fluorophores, and labeling strategies are discussed in detail in the commentary.

     
 
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Table of Contents

  • Basic Protocol 1: Postsynthetic Labeling of Amino‐ or Thiol‐Modified RNA
  • Support Protocol 1: Mild Deprotection of Standard RNA Oligonucleotides with NH4OH/Ethanol and Triethylamine Trihydrofluoride
  • Support Protocol 2: Mild Deprotection of 2′‐ACE‐Protected RNA Oligonucleotides with Acetic Acid
  • Support Protocol 3: Gel Purification of RNA Oligonucleotides
  • Support Protocol 4: C8 Reversed‐Phase Purification of RNA Oligonucleotides
  • Basic Protocol 2: Data Acquisition and Analysis for Steady‐State Fret
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Postsynthetic Labeling of Amino‐ or Thiol‐Modified RNA

  Materials
  • Gel‐ and HPLC‐purified RNA sample with amino or thiol functionality (see protocol 5)
  • Chloroform, buffered (see recipe)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% and 80% (v/v) ethanol
  • Succinimidyl ester (for amino‐modified RNA) or maleimide derivative (for thiol‐modified RNA) of fluorophore of choice (e.g., Molecular Probes, Sigma‐Aldrich, Amersham Pharmacia Biotech; for selection of fluorophores, see )
  • Dimethylsulfoxide (DMSO), anhydrous (e.g., Fisher)
  • 100 mM sodium tetraborate, pH 8.5 (for amino‐modified RNA, see recipe) or 100 mM HEPES‐KOH, pH 7.0 (for thiol‐modified RNA, see recipe)
  • 100 mM ATP or GTP ( appendix 2A; recipe for dNTPs)
  • Speedvac evaporator (e.g., Savant)
  • Aluminum foil
  • Tube shaker (e.g., Fisher)

Support Protocol 1: Mild Deprotection of Standard RNA Oligonucleotides with NH4OH/Ethanol and Triethylamine Trihydrofluoride

  Materials
  • RNA oligonucleotide attached to solid synthesis support (1‐µmol scale), made using standard β‐cyanoethyl phosphoramidites (see above for suggested suppliers and unit 3.5 for synthesis protocols)
  • 29% (v/v) ammonium hydroxide (e.g., Fisher)
  • 100% and 80% (v/v) ethanol (e.g., Fisher)
  • Triethylamine trihydrofluoride (Aldrich or Acros)
  • N,N‐Dimethylformamide (e.g., Fisher; optional)
  • 1‐Butanol (e.g., Fisher)
  • 1.7‐mL screw‐top tube (e.g., Eppendorf Safe‐Twist)
  • Parafilm (e.g., Fisher)
  • Aluminum foil
  • Heating block (e.g., Fisher)
  • 14‐mL Falcon centrifuge tube (e.g., Fisher)
  • Speedvac evaporator (e.g., Savant)
  • Tube shaker (e.g., Fisher)

Support Protocol 2: Mild Deprotection of 2′‐ACE‐Protected RNA Oligonucleotides with Acetic Acid

  • RNA oligonucleotide made using 5′‐silyl‐2′‐orthoester protection chemistry, already cleaved from solid support and base deprotected (Dharmacon Research)
  • 100 mM acetic acid/TEMED, pH 3.8 (see recipe)

Support Protocol 3: Gel Purification of RNA Oligonucleotides

  Materials
  • Urea (e.g., Fisher)
  • 38% (w/v) acrylamide/2% (w/v) bisacrylamide (e.g., Fisher; unit 10.4)
  • 10× TBE electrophoresis buffer ( appendix 2A)
  • 50% (w/v) APS (see recipe)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED; Fisher)
  • Deprotected RNA sample (see protocol 2Support Protocol 1 or protocol 32)
  • 2× formamide loading buffer (see recipe)
  • Elution buffer (see recipe)
  • Chloroform, buffered (see recipe)
  • 100 mM ATP or GTP ( appendix 2A; recipe for dNTPs)
  • 100% and 80% (v/v) ethanol
  • Vertical slab gel electrophoresis apparatus (e.g., 20 × 16–cm system from CBS Scientific), including glass plates, 1‐mm spacers, fitting seal, 1‐mm one‐ or two‐well comb, clamps, and aluminum plate
  • Power supply (e.g., Fisher)
  • 60‐mL syringe with bent 18‐G needle (e.g., Fisher)
  • Heating compartment (e.g., Fisher) filled with copper shot (e.g., Fisher), or other temperature‐controlled heating block, set at 95°C
  • Large‐volume gel‐loading pipet tips (e.g., Fisher)
  • Aluminum foil
  • Plastic wrap (e.g., Saran wrap)
  • 20 × 20–cm TLC plate with fluorescent indicator (e.g., Fisher)
  • 312‐ or 254‐nm hand‐held UV lamp (e.g., Fisher)
  • Empty Poly‐Prep chromatography column (Bio‐Rad)
  • Tube shaker (e.g., Fisher)
  • 14‐mL Falcon centrifuge tube (e.g., Fisher)
  • Speedvac evaporator (e.g., Savant)

Support Protocol 4: C8 Reversed‐Phase Purification of RNA Oligonucleotides

  Materials
  • Gel‐purified RNA sample (see protocol 4)
  • 100 mM TEAA buffer, pH 7 (see recipe)
  • Acetonitrile (see recipe)
  • Centrifugal filtration unit (0.45‐µm; Amicon)
  • HPLC system (unit 10.5) with 4.6 × 250–mm Microsorb 100 C8 analytical column (5‐µm particle size; Varian) and optional guard column
  • Speedvac evaporator (e.g., Savant)
  • Aluminum foil
  • Spectrophotometer (220 to 800 nm)
  • Additional reagents and equipment for reversed‐phase HPLC (unit 10.5)

Basic Protocol 2: Data Acquisition and Analysis for Steady‐State Fret

  Materials
  • Fluorophore‐labeled RNA sample of choice (see protocol 1)
  • Buffer of choice
  • Argon gas (optional)
  • Contrad 70 detergent (e.g., Fisher)
  • Water or oil pump connected to side‐arm Erlenmeyer flask (optional)
  • Luer‐tip syringe (e.g., Fisher)
  • Centrifugal filtration unit (0.45‐µm; Amicon)
  • Quartz microcuvette (fill volume 120 to 150 µL; e.g., Starna)
  • Research‐type spectrofluorometer (e.g., Thermo Spectronic AMINCO‐Bowman Series 2 or equivalent instrument from Jobin Yvon, Hitachi, or others), ideally with temperature control and stopped‐flow equipment for fast kinetics
  • Large‐volume gel‐loading tips (e.g., Fisher)
NOTE: If a circulating water bath is used with the spectrofluorometer, the temperature difference between the bath and the cuvette content should be calibrated.
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Figures

Videos

Literature Cited

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