5′‐Iodination of Solid‐Phase‐Linked Oligodeoxyribonucleotides

Eric T. Kool1, Gregory P. Miller1

1 Stanford University, Stanford, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.19
DOI:  10.1002/0471142700.nc0419s14
Online Posting Date:  November, 2003
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Abstract

5′‐Iodinated oligodeoxyribonucleotides readily react with 3′‐phosphorothioated DNA in the presence of a complementary template to yield a conjugate that is identical to natural DNA in every respect except that one oxygen atom in the phosphodiester backbone is replaced by a sulfur atom. The 5′‐iodo group is easily converted to a variety of other functional groups and will quickly react with thiol‐containing labels to yield stable thioether conjugates. This unit presents manual and automated procedures for converting the 5′‐hydroxyl of protected CPG–bound oligodeoxyribonucleotides to an iodo group and for releasing and purifying the products.

Keywords: iodination; 5′‐modification; solid‐phase; oligodeoxyribonucleotide; electrophile; ligation

     
 
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Table of Contents

  • Basic Protocol 1: Manual Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support
  • Alternate Protocol 1: Automated Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Manual Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support

  Materials
  • Anhydrous N,N‐dimethylformamide (DMF)
  • Iodination solution (see recipe)
  • Methylene chloride (CH 2Cl 2)
  • 28% (v/v) ammonium hydroxide (NH 4OH)
  • DNA synthesizer (e.g., ABI; see appendix 3C)
  • 0.2 to 10 µmol DNA synthesis column with long‐chain alkylamine controlled‐pore glass (CPG) support
  • 1‐ and 10‐mL syringes
  • Shaker, rocker, or other agitating device
  • C18 reversed‐phase HPLC column (unit 10.5)
  • Speedvac evaporator
  • Additional reagents and equipment for oligonucleotide synthesis (Chapters 3 and 4 and appendix 3C) and reversed‐phase HPLC (unit 10.5)

Alternate Protocol 1: Automated Procedure for 5′‐Iodination of Oligodeoxyribonucleotides on a Solid Support

  • Glass wool or line filter (e.g., preparation and delivery line filter from ABI)
  • Empty DNA synthesis column (e.g., 1000‐Å CPG column; ABI)
  • 10‐mL syringes
  • Clean, oven‐dried phosphoramidite bottle compatible with synthesizer
  • Reagent bottle compatible with synthesizer, filled with anhydrous DMF
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Figures

Videos

Literature Cited

   Dimitrijevich, S.D., Verheyden, J.P.H., and Moffatt, J.G. 1979. Halo sugar nucleosides. 6. Synthesis of some 5′‐deoxy‐5′‐iodo and 4′,5′‐unsaturated purine nucleosides. J. Org. Chem. 44:400‐406.
   Miller, G.P. and Kool, E.T. 2002. A simple method for electrophilic functionalization of DNA. Org. Lett. 4:3599‐3601.
   Xu, Y. and Kool, E.T. 1997. A novel 5′‐iodonucleoside allows efficient non‐enzymatic ligation of single‐stranded and duplex DNAs. Tetrahedron Lett. 38:5595‐5598.
   Xu, Y. and Kool, E.T. 1998. Chemical and enzymatic properties of bridging 5′‐S‐phosphorothioester linkages in DNA. Nucl. Acids Res. 26:3159‐3164.
Key References
   Miller and Kool, 2002. See above.
  Reports a method to iodinate the 5′‐carbon of oligo‐deoxyribonucleotides on a solid support.
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