Rapid Magnesium Chelation as a Method to Study Real‐Time Tertiary Unfolding of RNA

Emily J. Maglott1, Gary D. Glick1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.7
DOI:  10.1002/0471142700.nc1107s06
Online Posting Date:  November, 2001
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This unit describes a method to measure the unfolding of RNA tertiary structure on a millisecond time scale. A stopped‐flow spectrophotometer is used to measure the rate of unfolding induced by the addition of EDTA to an RNA whose tertiary structure has been stabilized in the presence of magnesium ions. Using this methodology, rate constants for unfolding of tertiary or secondary structure can be obtained over a range of temperatures, and these values can be used to construct Arrhenius and Eyring plots, from which activation energy, Arrhenius pre‐exponential factor, and enthalpy and entropy of activation can be obtained. These data provide information about the energy of the transition state and the energy barriers between secondary and tertiary structure, which is necessary for predicting RNA tertiary structure from secondary structure.

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Table of Contents

  • Basic Protocol 1: Measurement of Unfolding Rates of RNA Tertiary Structure
  • Support Protocol 1: Folding and Equilibration of RNA Samples
  • Support Protocol 2: Flow Dialysis of RNASamples
  • Support Protocol 3: Determination of Tertiary Unfolding Rates and Activation Parameters
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: Measurement of Unfolding Rates of RNA Tertiary Structure

  • EDTA buffer (see recipe) at EDTA concentration needed to chelate all Mg2+ in RNA sample
  • Mg2+ buffer (see recipe)
  • Folded RNA sample (see protocol 2)
  • Syringe filters, 0.22‐µm
  • 10‐mL and 3‐mL luer‐tip syringes (e.g., Fisher)
  • Parafilm
  • Stopped‐flow spectrophotometer (e.g., Applied Photophysics SX18.MV or equivalent instruments from Olis Instruments or Hi‐Tech Scientific)
  • Side‐arm Erlenmeyer flask
  • Additional reagents and equipment for folding RNA (see protocol 2) and analyzing kinetic traces to determine unfolding rates and activation parameters (see protocol 4)

Support Protocol 1: Folding and Equilibration of RNA Samples

  • Dialyzed RNA sample (see protocol 3)
  • Mg2+ buffer (see recipe)
  • 75°C water bath
  • Spectrophotometer

Support Protocol 2: Flow Dialysis of RNASamples

  • Mg2+ buffer (see recipe)
  • Argon source
  • Dry RNA sample of interest
  • Bottle‐top filtration apparatus containing a 0.22‐µm filter (e.g., Corning)
  • 10‐well microdialyzer (Spectrum)
  • Peristaltic pump
  • Cellulose ester membrane for use with 10‐well microdialyzer (Spectrum), MWCO 5000

Support Protocol 3: Determination of Tertiary Unfolding Rates and Activation Parameters

  • Graphical analysis software (e.g., Kaleidagraph from Synergy Software)
  • Statistical analysis software (e.g., SAS from SAS Institute)
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Literature Cited

Literature Cited
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