Characterization of Tertiary Folding of RNA by Circular Dichroism and Urea

Tobin R. Sosnick1

1 University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 11.5
DOI:  10.1002/0471142700.nc1105s04
Online Posting Date:  May, 2001
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Abstract

CD spectroscopy can be used to monitor RNA tertiary folding transitions that may not be observable by absorbance spectroscopy. With the use of computer‐controlled titrators, data can be acquired rapidly, and accurate thermodynamic properties can be obtained over a wide variety of conditions. Thus, CD spectroscopy provides a useful complement to site‐resolved or chemical modification methods.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of a Circular Dichroism (CD) Spectrum
  • Basic Protocol 2: Measurement and Analysis of Mg2+‐Induced Folding Transitions
  • Basic Protocol 3: Measurement and Analysis of a Urea Titration
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of a Circular Dichroism (CD) Spectrum

  Materials
  • Unfolded RNA sample
  • UV‐VIS spectrophotometer
  • 1‐cm‐path‐length quartz cuvette
  • 90°C water bath
  • Circular dichroism (CD) spectrometer capable of far‐UV measurements (e.g., Jasco or AVIV Associates)
  • Magnetic stir bar to fit cuvette
  • Additional reagents and equipment for RNA renaturation (unit 6.3)

Basic Protocol 2: Measurement and Analysis of Mg2+‐Induced Folding Transitions

  Materials
  • Unfolded RNA sample
  • 10 mM and 1 M magnesium stock solutions (ultrapure, autoclaved)
  • Plastic capillary tubing
  • Calibrated gas‐tight glass syringe
  • Magnetic stir bar to fit cuvette
  • Additional reagents and equipment for determining CD spectrum (see protocol 1)

Basic Protocol 3: Measurement and Analysis of a Urea Titration

  Materials
  • Unfolded RNA sample
  • Urea (ultrapure; filter through 0.2‐µm filter)
  • Additional reagents and equipment for determining CD spectrum (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
   Banerjee, A.R. and Turner, D.H. 1995. The time dependence of chemical modification reveals slow steps in the folding of a group I ribozyme. Biochemistry 34:6504‐6512.
   Fang, X., Pan, T., and Sosnick, T.R. 1999. A thermodynamic framework and cooperativity in the tertiary folding of a Mg2+‐dependent ribozyme. Biochemistry 38:16840‐16846.
   Makhatadze, G.I. and Privalov, P.L. 1992. Protein interactions with urea and guanidinium chloride. A calorimetric study. J. Mol. Biol 226:491‐505.
   Myers, J.K., Pace, C.N., and Scholtz, J.M. 1995. Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding [published erratum appears in Protein. Sci. 1996 May;5(5):981]. Protein Sci. 4:2138‐2148.
   Pan, T. and Sosnick, T.R. 1997. Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity. Nat. Struct. Biol. 4:931‐938.
   Sclavi, B., Woodson, S., Sullivan, M., Chance, M.R., and Brenowitz, M. 1997. Time‐resolved synchrotron X‐ray “footprinting,” a new approach to the study of nucleic acid structure and function: Application to protein‐DNA interactions and RNA folding. J. Mol. Biol. 266:144‐159.
   Shelton, V., Sosnick, T.R., and Pan, T. 1999. Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding. Biochemistry 38:16831‐16839.
   Zarrinkar, P.P. and Williamson, J.R. 1994. Kinetic intermediates in RNA folding. Science 265:918‐924.
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