Probing RNA Structures with Hydroxyl Radicals

Daniel W. Celander1

1 Loyola University Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.5
DOI:  10.1002/0471142700.nc0605s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Fe(II)‐EDTA can be used to conveniently generate hydroxyl radicals to promote cleavage of RNA at nucleotide resolution. Two procedures are described, involving the generation of free radicals from solvated molecular oxygen and from hydrogen peroxide added to the RNA solution. Unlike other footprinting reagents, hydroxyl radicals cleave the sugar‐phosphate backbone at every residue and thus provide uniform cleavage in a given RNA secondary structure. Because some positions become protected by tertiary folding, this reagent is useful for monitoring the global folding of RNA at equilibrium.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Strand Scission of RNA Using O2‐Derived Free Radicals
  • Alternate Protocol 1: Strand Scission of RNA Using H2O2‐Derived Free Radicals
  • Commentary
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Strand Scission of RNA Using O2‐Derived Free Radicals

  Materials
  • End‐labeled RNA with a specific activity ≥2 × 106 dpm/pmol (unit 6.1)
  • Appropriate buffers and RNA‐binding ligands (see )
  • 1,4‐Dithiothreitol (DTT; store at ‐20°C)
  • (NH 4) 2Fe(II)(SO 4) 2⋅6H 2O powder (store at room temperature)
  • 200 mM Na 2EDTA, pH 8.0 ( appendix 2A), prepared in sterile water
  • 100 mM thiourea in sterile water (store at 4°C)
  • 2× urea loading buffer ( appendix 2A)
  • T1 nuclease digest and alkaline hydrolysis of end‐labeled RNA (unit 6.1)
  • Distilled, deionized water, autoclaved before use
  • 0.5‐ and 1.5‐mL polypropylene microcentrifuge tubes, sterile
  • RNase‐free micropipettors and tips
  • Water bath or suitable incubator
  • Radioanalytic detection instrument or autoradiography film
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; see appendix 3B)

Alternate Protocol 1: Strand Scission of RNA Using H2O2‐Derived Free Radicals

  • 0.6% (w/w) hydrogen peroxide (H 2O 2) in sterile water, freshly prepared from 30% (w/w) commercial solution
  • Sodium ascorbate powder
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Cate, J.H., Gooding, A.R., Podell, E., Zhou, K., Golden, B.L., Kundrot, C.E., Cech, T.R., and Doudna, J.A. 1996. Crystal structure of a group I ribozyme domain: principles of RNA packing. Science 273:1678‐1685.
   Celander, D.W. and Cech, T.R. 1990. Iron(II)‐ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: Similar reactivity toward single‐ and double‐stranded forms. Biochemistry 29:1355‐1361.
   Celander, D.W. and Cech, T.R. 1991. Visualizing the higher order folding of a catalytic RNA molecule. Science 251:401‐407.
   Darsillo, P. and Huber, P.W. 1991. The use of chemical nucleases to analyze RNA‐protein interactions. The TFIIIA‐5 S rRNA complex. J. Biol. Chem. 266:21075‐21082.
   Hertzberg, R.P. and Dervan, P.B. 1984. Cleavage of DNA with methidiumpropyl‐EDTA‐iron(II): Reaction conditions and product analyses. Biochemistry 23:3934‐3945.
   Heuer, T.S., Chandry, P.S., Belfort, M., Celander, D.W., and Cech, T.R. 1991. Folding of group I introns from bacteriophage T4 involves internalization of the catalytic core. Proc. Natl. Acad. Sci. U.S.A. 88:11105‐11109.
   Latham, J.A. and Cech, T.R. 1989. Defining the inside and outside of a catalytic RNA molecule. Science 245:276‐282.
   Pan, T. 1995. Higher order folding and domain analysis of the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry 34:902‐909.
   Rosenstein, S.P. and Been, M.D. 1996. Hepatitis delta virus ribozymes fold to generate a solvent‐inaccessible core with essential nucleotides near the cleavage site phosphate. Biochemistry 35:11403‐11413.
   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.
   Sclavi, B., Sullivan, M., Chance, M.R., Brenowitz, M., and Woodson, S.A. 1998. RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting. Science 279:1940‐1943.
   Westhof, E., Wesolowski, D., and Altman, S. 1996. Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)‐EDTA reagent. J. Mol. Biol. 258:600‐613.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library