Engineering Disulfide Cross‐Links in RNA Using Thiol‐Disulfide Interchange Chemistry

Scott B. Cohen1, Thomas R. Cech1

1 University of Colorado, Boulder, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.1
DOI:  10.1002/0471142700.nc0501s00
Online Posting Date:  May, 2001
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Abstract

Protocols for postsynthetic modification of 2‐amino‐containing oligoribonucleotides with either an alkyl‐phenyl disulfide or an alkyl thiol group are described. These groups react under mild conditions to form disulfide cross‐links by thiol‐disulfide interchange. These reactants do not form a disulfide bond when incorporated on opposite faces of a short continuous RNA helix, but do form disulfide bonds rapidly when they are placed in proximity. In addition, by incorporating these groups at various positions on large RNAs by semisynthesis, the dynamics of thermal motions can be detected. Such motions are believed to be linked to biological function, and the protocols presented in this unit are among the few simple ways to assess such dynamics.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of RNA Oligonucleotides Containing a Site‐Specific 2′ Amine Group
  • Basic Protocol 2: Preparation of 32P‐Labeled RNA Containing an Alkyl Phenyl Disulfide Group
  • Basic Protocol 3: Preparation of RNA Containing Alkyl Thiol Group
  • Basic Protocol 4: Cross‐Linking of RNA Through Thiol‐Disulfide Interchange
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of RNA Oligonucleotides Containing a Site‐Specific 2′ Amine Group

  Materials
  • Solid support–bound product of automated RNA synthesis (1‐µmol synthesis scale)
  • 3:1 (v/v) concentrated ammonium hydroxide (NH 4OH)/absolute ethanol
  • 24:46:30 (v/v/v) triethylamine/1‐methyl‐2‐pyrrolidinone/triethylamine trihydrofluoride (see recipe)
  • TE buffer, pH 7.5 ( appendix 2A)
  • NAP‐25 Sephadex column (Amersham Pharmacia Biotech)
  • 1 M sodium chloride
  • Absolute ethanol
  • TBE buffer ( appendix 2A)
  • 80% formamide/TBE solution (see recipe)
  • Denaturing polyacrylamide gel: 20% polyacrylamide/8 M urea in TBE buffer, dimensions 20 cm long × 26 cm wide × 0.3 cm thick (see appendix 3B, CPMB UNIT or Sambrook et al., )
  • TEN buffer (see recipe)
  • 4‐mL screw‐cap vial
  • Teflon tape
  • Water baths, 55° and 65°C
  • Rotary drying system for microcentrifuge tubes (Savant)
  • 40‐mL Oak Ridge centrifuge tube
  • Preparative centrifuge (Sorvall or Beckman)
  • UV lamp (hand held)
  • 50‐mL polypropylene centrifuge tube
  • 0.45‐µm cellulose acetate filter
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis and UV shadowing ( appendix 3B, CPMB UNIT or Sambrook et al., )

Basic Protocol 2: Preparation of 32P‐Labeled RNA Containing an Alkyl Phenyl Disulfide Group

  Materials
  • RNA oligonucleotide with 2′ amine group (100 µM in TE buffer; see protocol 1)
  • ≥0.1 Ci/µL [γ‐32P]ATP (6000 Ci/mmol)
  • 10 U/µL T4 polynucleotide kinase and 10× buffer (New England Biolabs)
  • 1 M sodium chloride
  • Absolute ethanol
  • 1 M sodium borate buffer, pH 8
  • 500 mM N‐succinimidyl‐3‐(2‐pyridyldithio) propionate (S.3; Pierce Chemicals) in N,N‐dimethylformamide (prepare solution just before use)
  • 70 mM thiophenol in absolute ethanol
  • TE buffer, pH 7.5 ( appendix 2A)
  • 1× TBE buffer ( appendix 2A)
  • 80% formamide/TBE solution (see recipe)
  • Denaturing polyacrylamide gel: 20% polyacrylamide/8 M urea in TBE buffer, dimensions 20 cm long × 10 cm wide × 0.05 cm thick (see appendix 3B, CPMB UNIT or Sambrook et al., )
  • TEN buffer (see recipe)
  • 10 mM sodium acetate buffer, pH 4.5 ( appendix 2A)
  • Preparative centrifuge (Sorvall or Beckman)
  • X‐ray film for autoradiography
  • Water bath, 37°C
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3B, CPMB UNIT or Sambrook et al., )
CAUTION: Step should be performed in a fume hood because of the stench of thiophenol. Any labware that comes in contact with thiophenol should be soaked in bleach solution.

Basic Protocol 3: Preparation of RNA Containing Alkyl Thiol Group

  Materials
  • 20 nmol RNA oligonucleotide with 2′ amine group (see protocol 1)
  • 1 M sodium borate buffer, pH 8
  • 1 M sodium chloride
  • 500 mM N‐succinimidyl‐3‐(2‐pyridyldithio) propionate (S.3; Pierce Chemicals) in N,N‐dimethylformamide (prepare just before use)
  • Absolute ethanol
  • 1 M dithiothreitol (DTT) in water ( appendix 2A)
  • TEN buffer (see recipe)
  • TE buffer, pH 7.5 ( appendix 2A)
  • Water bath, 37°C

Basic Protocol 4: Cross‐Linking of RNA Through Thiol‐Disulfide Interchange

  Materials
  • 32P‐labeled RNA modified with phenyl disulfide S.1 (≥105 cpm/µL in 10 mM sodium acetate buffer, pH 4.5; see protocol 2)
  • RNA modified with alkyl thiol S.2 (1 µM in TE buffer; see protocol 3)
  • 100 mM sodium acetate buffer, pH 4.5 ( appendix 2A)
  • 100 mM magnesium chloride
  • 5 M sodium chloride
  • Formamide quenching solution (see recipe)
  • 1 M sodium HEPES buffer, pH 7.5 ( appendix 2A)
  • 0.65‐mL microcentrifuge tube
  • Water bath, 30°C
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis ( appendix 3B, CPMB UNIT or Sambrook et al., )
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Figures

Videos

Literature Cited

Literature Cited
   Benseler, F., Williams, D.M., and Eckstein, F. 1992. Synthesis of suitably‐ protected phosphoram‐ idites of 2′ ‐ fluoro‐ 2′ ‐ deoxyguanosine and 2′ ‐ amino‐ 2′ ‐ deoxyguanosine for incorporation into oligoribonucleotides. Nucleosides Nucleotides 11:1333‐1351.
   Chen, C.B. and Sigman, D.S. 1988. Sequence‐ specific scission of RNA by 1,10‐ phenanthroline‐ copper linked to deoxyoligonucleotides. J. Am. Chem. Soc. 110:6570‐6572.
   Cohen, S.B. and Cech, T.R. 1997. Dynamics of thermal motions within a large catalytic RNA investigated by cross‐linking with thiol‐disulfide interchange. J. Am. Chem. Soc. 119:6259‐6268.
   Goodwin, J.T., Osborne, S.E., Scholle, E.J., and Glick, G.D. 1996. Design, synthesis, and analysis of yeast tRNAPhe analogs possessing intra‐ and interhelical disulfide cross‐links. J. Am. Chem. Soc. 118:5207‐5215.
   Imazawa, M. and Eckstein, F. 1979. Facile synthesis of 2′‐amino‐2′‐deoxyribo‐furanosyl purines. J. Org. Chem. 44:2039‐2041.
   Moore, M.J. and Sharp, P.A. 1992. Site‐specific modification of pre‐mRNA: the 2′‐hydroxyl groups at the splice sites. Science 256:992‐997.
   Patai, S. 1974. The Chemistry of the Thiol Group. John Wiley & Sons, New York, N.Y.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
   Sigurdsson, S.T., Tuschl, T., and Eckstein, F. 1995. Probing RNA tertiary structure: Interhelical cross‐linking of the hammerhead ribozyme. RNA 1:575‐583.
   Verheyden, J.P.H., Wagner, D., and Moffatt, J.G. 1971. Synthesis of some pyrimidine 2′‐amino‐2′‐deoxynucleosides. J. Org. Chem. 36:250‐254.
   Wincott, F., DiRenzo, A., Shaffer, C., Grimm, S., Tracz, D., Workman, C., Sweedler, D., Gonzalez, C., Scaringe, S., and Usman, N. 1995. Synthesis, deprotection, analysis. and purification of RNA and ribozymes. Nucl. Acids Res. 23:2677‐2684.
Key References
   Cohen and Cech, 1997. See above.
  Describes the initial development of thiol‐disulfide interchange chemistry and its application to measuring conformational dynamics within a 310‐nt catalytic RNA.
   Sigurdsson et al., 1995. See above.
  Presents the first example of the use of chemistry derived from a 2′ amine to tag the RNA backbone in a sequence‐specific fashion.
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